Regulation of transcription through ctcf loop anchors

ABSTRACT

Disclosed are methods of altering expression of a gene with a promoter region CTCF binding site. Also disclosed are compositions and methods useful for treating a disease or condition involving over-expression or under-expression of a gene with a promoter region CTCF binding site. Further disclosed are cells and non-human animals with modified a promoter region CTCF binding site, as well as methods for screening for compounds that can modify the expression of a gene with a promoter region CTCF binding site.

RELATED APPLICATION

This application is a national stage filing under 35 U.S.C. 371 of International Application No. PCT/US2017/065918, filed Dec. 12, 2017, which claims the benefit of U.S. Provisional Application Ser. No. 62/433,234, filed Dec. 12, 2016, the contents of which are hereby incorporated by reference in their entirety. International Application No. PCT/2017/065918 was published under PCT Article 21(2) in English.

GOVERNMENT SUPPORT

This invention was made with government support under Grant Nos. HG002668 and CA109901 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The ability to activate transcription of specific genes is fundamental to the establishment of gene expression programs that define cell identity. To accomplish this, transcription factors (TFs) bind enhancer elements and regulate transcription from the promoters of nearby or distant genes through physical contacts that involve looping of DNA between enhancers and promoters (Bonev and Cavalli, 2016; Buecker and Wysocka, 2012; Bulger and Groudine, 2011; Fraser et al., 2015; Heard and Bickmore, 2007; de Laat and Duboule, 2013; Müller et al., 1989; Pombo and Dillon, 2015; Spitz, 2016; Tolhuis et al., 2002). However, the mechanisms that ensure that specific enhancers interact with specific promoters are not well understood. Most study of eukaryotic enhancer-promoter interactions has focused on cofactors that lack DNA binding capabilities and bridge enhancer-bound transcription factors and the promoter-bound transcription apparatus (Allen and Taatjes, 2015; Deng et al., 2012; Jeronimo et al., 2016; Kagey et al., 2010; Malik and Roeder, 2010, 2016; Petrenko et al., 2016; Phillips-Cremins et al., 2013). Some enhancer-promoter interactions are likely determined by the nature of transcription factors bound at the two sites (Muerdter and Stark, 2016), but there is little understanding of the rules that govern these interactions.

Recent studies have revealed that specific chromosome structures play important roles in gene control. Enhancer-promoter interactions generally occur within larger chromosomal loop structures formed by the interaction of CTCF proteins bound to each of the loop anchors (Dekker and Mirny, 2016; Fraser et al., 2015; Gibcus and Dekker, 2013; Gorkin et al., 2014; Hnisz et al., 2016; Merkenschlager and Nora, 2016). These loop structures, variously called TADs, loop domains, CTCF contact domains and insulated neighborhoods, tend to insulate enhancers and genes within the CTCF-CTCF loops from elements outside those loops (Dixon et al., 2012, 2016; Dowen et al., 2014; Franke et al., 2016; Hnisz et al., 2016; Ji et al., 2016; Lupiáñez et al., 2015; Narendra et al., 2015; Nora et al., 2012; Phillips-Cremins et al., 2013; Rao et al., 2014; Tang et al., 2015). Constraining DNA interactions within CTCF-CTCF loop structures in this manner may facilitate proper enhancer-promoter contacts.

SUMMARY OF THE INVENTION

CTCF does not generally occupy enhancer and promoter elements (Cuddapah et al., 2009; Dixon et al., 2012; Handoko et al., 2011; Ji et al., 2016; Kim et al., 2007; Parelho et al., 2008; Phillips-Cremins et al., 2013; Rao et al., 2014; Rubio et al., 2008; Tang et al., 2015; Wendt et al., 2008), but where CTCF does bind these elements, it may engender enhancer-promoter interactions (Guo et al., 2015; Lee et al., 2017; Splinter et al., 2006; de Wit et al., 2015). This consideration led us to further investigate the class of human genes that contain CTCF-bound sites at promoters to learn whether these are utilized to facilitate contacts with enhancers via CTCF-CTCF interactions. We report here that 2000 human genes have highly conserved promoter-proximal sites that are bound by CTCF regardless of the cell type examined and that these sites can form contacts with diverse cell-type specific enhancers. These genes appear to have evolved CTCF enhancer-docking sites in order to facilitate contacts with the diverse CTCF-bound enhancers formed by cell-type specific transcription factors during development, and thus experience activation in a broad range of cell types. Interestingly, this set of genes with CTCF-bound enhancer-docking sites includes many important cancer-associated genes, and the enhancer-docking site of one of these, MYC, was studied in detail.

Elevated expression of the c-MYC transcription factor occurs frequently in human cancers and is associated with tumor aggression and poor clinical outcome (Berns et al., 2013; Grotzer et al, 2001; Nesbit et al., 1999; Rao et al., 1998; Dang, 2012). There has been considerable interest in understanding the mechanisms responsible for aberrant transcriptional regulation of MYC in tumor cells. Promoter-proximal regulatory sequences were identified in early studies, but these were not sufficient to recapitulate endogenous patterns of MYC expression (Lavenu et al., 1994; Wierstra, 2008). Subsequent reports noted putative regulatory elements that occur over 1 megabase (Mb) away from MYC, suggesting that distal elements might be involved in MYC regulation (Ahmadiyeh et al., 2010; Hallikas et al., 2006; Pomerantz et al., 2009; Sotelo et al., 2010; Tuupanen et al., 2009; Wright et al., 2010; Yochum et al., 2008). Recent studies have described large tumor-specific super-enhancers in the 3 Mb region surrounding the MYC gene (Chapuy et al, 2013; Herranz et al., 2014; Hnisz et al., 2013; Lin et al., 2016; Wang et al., 2015; Xiang et al., 2014; Zhang et al, 2015). It is not clear how these large enhancer clusters, which differ in size, composition and distance from MYC, all accomplish the same task of stimulating MYC overexpression in a broad spectrum of tumors. Molecular features common to these regulatory elements might prove to be valuable for therapeutic targeting in cancer.

Genetic and epigenetic perturbation of the MYC enhancer-docking site reduces CTCF binding, super-enhancer interaction, MYC gene expression and tumor cell proliferation. These observations reveal a mechanism for enhancer-promoter interaction that is employed during development to allow genes to have cell-specific contacts with diverse enhancers and is exploited by cancer cells to facilitate oncogenic expression of genes driven by diverse super-enhancers.

We show here that diverse tumor-specific super-enhancers acquired throughout the 3 Mb MYC insulated neighborhood functionally interact with a single conserved site containing densely clustered CTCF motifs in the MYC promoter. CRISPR-mediated deletion analysis shows that this common CTCF site is required for super-enhancer looping to the MYC promoter, high MYC expression and rapid cell proliferation in multiple cancers. Targeted methylation of the MYC enhancer anchor by dCAS9-DNMT3A-3L fusion proteins abrogate CTCF binding with consequent loss of MYC expression, suggesting a common vulnerability and a novel approach for therapeutic targeting. Disruption of CTCF protein binding with the MYC promoter CTCF binding site by compounds including targeted nucleic acid derivatives are predicted to have a similar effect.

Disclosed herein are methods of altering expression of a gene with a promoter region (i.e., within or proximal (e.g., within 2.5 kilobases of the transcription start site) to the promoter) CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) by altering the interaction or binding between CTCF protein and CTCF binding site(s) to a promoter region of the gene. In some aspects, the gene is MYC. Generally said alteration will comprise inhibiting or reducing the binding of CTCF protein and CTCF binding site in the promoter region of the gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1), thereby reducing or eliminating expression of the gene. However said alteration may alternatively comprise enhancing the binding of CTCF protein to CTCF binding site in the promoter region of the gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1), thereby enhancing expression. Methods of therapy corresponding to alteration of expression of the gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) are also encompassed.

Disclosed herein are compositions useful for treating a disease or condition involving over-expression or under-expression of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1), comprising a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity; and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site and/or a portion within 200-300 nucleotides of either side of the promoter region CTCF binding site. In some aspects, the disease or condition is cancer.

Also disclosed herein are compositions useful for treating a disease or condition involving over-expression or under-expression of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1). In some embodiments, the composition comprises a mixture of proteins (e.g., a fusion protein comprising a catalytically inactive site specific nuclease fused to an effector domain having methylation activity) and nucleic acid sequences (e.g., one or more guide RNAs, one or more sequences encoding guide RNA). In some embodiments, the composition comprises one or more guide sequences and one or more nucleic acids encoding a fusion protein comprising a catalytically inactive site specific nuclease fused to an effector domain having methylation activity. In some embodiments, the composition comprises one or more nucleic acids encoding a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity and one or more nucleic acids encoding guide sequences. In some embodiments, the guide sequences are homologous or complementary to at least a portion of the MYC promoter region CTCF binding site and/or a portion within 200-300 nucleotides upstream or downstream of the MYC promoter region CTCF binding site. In some embodiments, the guide sequences are homologous or complementary to at least a portion of a promoter region CTCF binding site listed in Table S1 and/or a portion within 200-300 nucleotides upstream or downstream of the promoter region CTCF binding site listed in Table S1. In some embodiments, the guide sequences are homologous or complementary to at least a portion of a promoter region CTCF binding site for TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, or CSNK1A1 and/or a portion within 200-300 nucleotides upstream or downstream of the promoter region CTCF binding site for TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, or CSNK1A1. In some aspects, the disease or condition is cancer.

In some aspects, the effector domain comprises DNMT3A-3L or DNMT3A-3L lacking the 5′ nuclear localization signal (NLS) domain. In some aspects, the catalytically inactive site specific nuclease is a catalytically inactive Cas (e.g., Cas9); in other aspects the catalytically inactive site specific nuclease is a catalytically inactive Cpf1. In some aspects, the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DNMT3A-3L or dCas9-DNMT3A-3L lacking the 5′ NLS.

Also disclosed herein are methods for modulating methylation of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) in a cell comprising introducing into the cell a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity; and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site, thereby methylating or demethylating the promoter region CTCF binding site. In some aspects, methylation of the promoter region CTCF binding site of the gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) is increased or decreased by about 2-fold, 2.5-fold, 2.7-fold, 3.0 fold, or 4.0 fold or more. In some aspects, methylation of the promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) is increased or decreased by at least one site, at least two sites, at least three sites, at least five sites, at least ten sites, at least fifteen sites, at least twenty sites, at least twenty-five sites, or more. In some aspects, methylation of CpG in the promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) is increased or decreased by at least one site, at least two sites, at least three sites, at least five sites, at least ten sites, at least fifteen sites, at least twenty sites, at least twenty-five sites, or more.

Also disclosed herein are methods of modulating the expression of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) in a subject in need thereof comprising introducing into the subject a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity; and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site, thereby modulating the expression of mRNA of the gene in cells of the subject. In some aspects, expression of the gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) is increased or decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some aspects, protein expression is increased or decreased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

In some aspects, the effector domain comprises DNMT3A-3L. In some aspects, the catalytically inactive site specific nuclease is a catalytically inactive Cas (e.g., Cas9). In some aspects, the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DNMT3A-3L or dCas9-DNMT3A-3L without the 5′ NLS.

In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 genomic sequences (e.g., regions of the MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S 1promoter region CTCF binding site) are modified in the cell. In some embodiments, the genomic sequences to be modified are CTCF binding motifs. The cell may be a stem cell, a neuron, a post-mitotic cell, or a fibroblast. In some aspects, the cell is a human cell or a mouse cell. In some aspects, the cell is a cancer cell.

In certain embodiments, the methods further comprise introducing the cell into a non-human mammal. The non-human mammal may be a mouse.

Also disclosed are isolated modified cells produced by the methods described herein.

Also disclosed herein are methods of treating a subject in need thereof, comprising administering to the subject a composition that suppresses, reduces or eliminates the binding of CTCF to a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1). In some aspects the subject is human. In some aspects, the subject has cancer. In some aspects the cancer is colorectal cancer, leukemia or breast cancer.

Also disclosed herein are methods of screening for a compound that modulates expression of a gene having a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) comprising contacting a cell with a test agent; and measuring methylation in the promoter region CTCF binding site, wherein the test agent is identified as a compound that modulates expression if the level of methylation of the promoter region CTCF binding site in the cell contacted with the test agent differs from the level of methylation of said promoter region CTCF binding site in a control cell not contacted with the test agent.

Also disclosed herein are methods of screening for a compound that modulates expression of a gene having a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) comprising contacting a cell with a test agent; and measuring binding between CTCF protein and CTCF binding site(s) within the promoter region, wherein the test agent is identified as a compound that modulates expression of the gene if the level of binding between CTCF protein and CTCF binding site(s) within the promoter region in the cell contacted with the test agent differs from the level of binding in a control cell not contacted with the test agent.

In some aspects, the test agent comprises a small molecule. In some aspects, the test agent comprises a nucleic acid. In some aspects, the compound is identified as an anti-cancer agent.

The above discussed, and many other features and attendant advantages of the present inventions will become better understood by reference to the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

FIGS. 1A-1E. Constitutive CTCF sites at promoters loop to differential enhancers. (A) The number of genes with a constitutive CTCF site near the promoter that occur within a constitutive insulated neighborhood and have differential enhancers within that insulated neighborhood. A neighborhood was considered constitutive if it was identified in at least two out of three datasets and a CTCF site was considered constitutive if it was present in all three cell types. Genome interaction data from this study, (Heidari et al., 2014; Hnisz et al., 2016). H3K27ac and CTCF ChIP-seq data from this study, (Encode Consortium, 2012; Frietze et al., 2012; Gertz et al., 2013; Hnisz et al., 2016) (B) The 4.5 Mb region surrounding the MYC gene. The 2.8 Mb TAD containing MYC and part of the two adjacent TADs are indicated with thick black horizontal lines. Super-enhancers (data from (Becket et al., 2016; Frietze et al., 2012; Lin et al., 2012; Pope et al., 2014; Wang et al., 2011)) are shown in colored boxes for a panel of tumor cell lines that express MYC. (C) Chromosome interaction data at the MYC locus. HCT-116 SMC1 HiChIP interactions with an ORIGAMI score of at least 0.9 and a minimum PET count of 9 are shown as purple arcs. The insulated neighborhood spanning interaction is shown in blue. MCF7 CTCF and PolII ChIA-PET: interactions from the PolII ChIA-PET are shown as purple arcs and the insulated neighborhood spanning interactions from the CTCF ChIA-PET are shown in blue. Data from ENCODE and (Li et al., 2012). K562 RAD21 ChIA-PET interactions with an ORIGAMI score of at least 0.9 are shown as purple arcs. The insulated neighborhood spanning interaction is shown in blue and has an ORIGAMI score of 0.44. Data from (Heidari et al., 2014). Jurkat SMC1 ChIA-PET interactions with an ORIGAMI score of at least 0.99 are shown as purple arcs and the insulated neighborhood spanning interactions are shown in blue. Data from (Hnisz et al., 2016). CTCF ChIP-seq peaks are depicted as purple, super-enhancers are depicted as red and typical enhancers as grey rectangles. Data from this study, (Hnisz et al., 2016; Pope et al., 2014). (D) CTCF ChIP-seq and SMC1 ChIA-PET read pileup at the MYC promoter. Purple tracks display CTCF ChIP-seq signal in the four cell lines from panels C. Light blue track displays the read counts from read pileup of Jurkat SMC1 ChIA-PET data showing that the majority of the ChIA-PET reads are found at the enhancer docking site. Blue bars indicate CpG islands. ChIP-seq read counts are shown in reads per million sequenced reads per basepair. ChIA-PET reads are shown as read counts per basepair. (E) The top panel depicts all putative CTCF binding motifs as blue arrows indicating the orientation of the motif. 100 vertebrate conservation from UCSC genome browser is depicted in the bottom panel. The motif depicted in dark blue occurs in the most conserved region and shows the best match with consensus CTCF motif (e.g., CCGCGNGGNGGCAG; SEQ ID NO: 21).

FIGS. 2A-2D. Perturbation of common MYC enhancer docking site reduces CTCF occupancy, and looping to super-enhancers. (A) Schematic representation of the proposed model for an enhancer-docking site upstream of the MYC gene. (B) CTCF ChIP-seq data for the MYC locus in wild type (K562) and enhancer loop-anchor deletion (ΔK562) cells (left panel). A 210 bp segment in the middle of the CTCF binding site was deleted through CRISPR/Cas9 genome editing. The MYCL locus is shown as a control (right panel). Read counts are shown in reads per million sequenced reads per basepair. (C) qPCR showing the MYC mRNA levels after deletion of the enhancer anchor site in K562 cells. p-value was generated with a Students T-test. Error bars represent the standard deviation of the mean from three biological replicates. (D) 4C analysis showing reduced looping of common loop-anchor to super-enhancers in 210 bp deletion cells (AK562) versus parental cells (K562). Blowup shows the 4C interactions for five K562 specific super-enhancers. The 4C viewpoint is situated 112 basepairs up-stream of the deleted loop-anchor region. Shading represents the 90% confidence interval based on three biological replicates. Reads are shown in reads per million sequenced reads per basepair.

FIGS. 3A-3C. Homozygous deletion of the core CTCF motif reduces MYC expression. (A) Schematic representation of the experiment. HCT-116, K562, Jurkat and MCF7 cells were transduced with a construct expressing MYC under a pGK promoter and selected for successful integration. These cells were then transiently transfected with plasmid carrying Cas9 and a gRNA targeting the CTCF binding motif. Positive cells were identified and selected using fluorescence assisted cell sorting (FACS). These cells were multiplied and clonal populations were characterized. (B) Top panel, shows the position weight matrix for the CTCF motif. Bottom panel shows sequences from the selected K562, HCT-116, Jurkat and MCF7 cell lines. For the aneuploid MCF7 cell line the two most common mutations are depicted. Sequence highlighted in blue is complementary to the gRNA sequence targeting the most prominent motif which is shown here in bold sequence. (C) qPCR showing endogenous MYC mRNA levels in K562, HCT-116, Jurkat and MCF7 cells that express exogenous MYC together with the Δ-CTCF deletion counterparts. p-value was generated with a Students T-test. Error bars represent the standard deviation of the mean from three biological replicates.

FIGS. 4A-4D. dCas9-mediated methylation of the enhancer-docking site reduces MYC expression in tumor cells. (A) Top panel shows CTCF ChIP-seq at the MYC gene region in HCT-116 cells. ChIP-seq reads are shown in reads per million sequenced reads per basepair. Bottom panels shows a blowup of the ˜700 bp region underneath the CTCF peak depicting the CTCF motifs (blue arrows) and the gRNAs (red rectangles) used to target dCas9-DNMT3A-3L to the enhancer-docking site. Lollipop symbols indicate the location of CpGs that are assayed for methylation levels in C. (B) Schematic representation of the experiment. HCT-116 or HEK293T cells were transfected with plasmids encoding the dCAS9-DNMT3A-3L, green fluorescent protein (GFP) and a gRNA together with a plasmid encoding 2 additional gRNAs. HCT-116 cells were isolated by FACS after two days and DNA and RNA were isolated. HEK293T cells did not require sorting due to high (˜80%) transfection efficiencies. (C) Methylation at MYC promoter loop-anchor site in untreated cells, cells transfected with dCas9-DNMT3A-3L alone or with dCas9-DNMT3A-3L in conjunction with the 5 indicated gRNAs. (D) qPCR analysis of MYC mRNA levels and fraction of methylated CpGs for untreated, transfected and control transfected cells. Error bars represent the standard deviation of the mean for three biological replicates.

FIGS. 5A-5B. MYC-proximal enhancer docking site is used during development and differentiation. (A) Promoter Hi-C interaction data and H3K27Ac ChIP-seq at the MYC TAD for cell types that represent different stages in hematopoietic development. The 2.9 Mb TAD containing MYC and part of the two adjacent TADs are indicated with thick black horizontal lines. Promoter Hi-C interactions are shown as purple colored arcs; the intensity of purple color reflects the confidence score from (Javierre et al., 2016). H3K27Ac ChIP-seq signal is shown, measured in reads per million sequenced reads per basepair (data from (Bernstein et al., 2010; Encode Consortium, 2012; Schmidt et al., 2016; Xu et al., 2012)). Super-enhancers are depicted as red rectangles and typical enhancers as grey rectangles. The relative level of MYC transcripts in the corresponding cell types are shown as boxplots in fragments per kilobase of exon per million sequenced reads (FPKM), expression data from the BLUEPRINT consortium, fetal thymus expression data from the ENCODE consortium. (B) CTCF ChIP-seq at the MYC gene region in HCT-116 cells and the average signal of whole genome bisulfite sequencing data for a panel of five cell types. Average percent methylation of each CpG in the region is represented as a blue bar. ChIP-seq reads are shown in reads per million sequenced reads per basepair.

FIGS. 6A-6B. Enhancer-docking sites at additional genes with prominent roles in cancer. (A) Examples of genes with enhancer docking sites (EDS) from the different cell types analyzed. CTCF ChIP-seq peaks are shown as purple rectangles, typical enhancer are shown as grey rectangles and super-enhancers are shown as red rectangles. HCT-116 HiChIP interactions are shown in purple for the TG1F1 locus. The insulated neighborhood interactions are shown in blue. K562 RAD21 ChIA-PET interactions with an ORIGAMI score of at least 0.9 and a minimum PET count of 30 are shown as purple arcs for the VEGFA locus. The insulated neighborhood spanning interaction is shown in blue. Data from (Heidari et al., 2014). Jurkat SMC1 ChIA-PET interactions with an ORIGAMI score of at least 0.97 are shown as purple arcs and the insulated neighborhood spanning interactions are shown in blue for the RUNX1 locus. Data from (Hnisz et al., 2016a). (B) Conservation analysis of the CTCF motifs in the constitutive CTCF bound elements in enhancer-docking sites. The mean 46-way PhastCons score of the highest JASPAR scoring motifs in constitutive CTCF peaks within EDSs and their flanking regions are shown.

FIGS. 7A-7E. (A) The 4.5 Mb region surrounding the MYC gene. The 2.8 Mb TAD containing MYC and part of the two adjacent TADs are indicated with thick black horizontal lines. The TAD-spanning CTCF-CTCF loop is indicated in light blue. H3K27Ac ChIP-seq signal (reads per million sequenced reads per basepair, data from (Becket et al., 2016; Frietze et al., 2012; Lin et al., 2012; Pope et al., 2014; Wang et al., 2011)) is shown in dark blue for a panel of tumor cell lines that express MYC. Tumor super-enhancers in the MYC TAD are depicted as red and typical enhancers are depicted as grey rectangles. (B) Heatmap of the ORIGAMI processed HiChIP, unfiltered data showing the MYC TAD with flanking regions (chr8:127100000-131525000) and Heatmaps of Hi-C interaction data showing the MYC TAD with flanking regions (chr8:127100000-131525000) across seven different cell types (data from (Rao et al., 2014)). No effort was made so smooth the HiChIP data as opposed to the smoothened HiC data. Scale bars represent the contrast settings used, numbers indicate the maximum intensity cutoff. The color intensity represents the PET count and the cutoff is represented in PET numbers for the HiChIP data. (C) CTCF ChIP-seq across a panel of tumor cell lines (data from (Anders et al., 2014; Encode Consortium, 2012; Hnisz et al., 2016; Pope et al., 2014; Wang et al., 2012; Yan et al., 2013)), and from mouse T-helper cells and Opossum, Dog, and Rhesus macaque liver (data from (Schmidt et al., 2012; Stadler et al., 2011)). Read counts are shown in reads per million sequenced reads per basepair. (D) ChIA-PET read pileups at the MYC promoter and quantification of the reads in the three CTCF peaks indicated. Light blue tracks display the read counts from read pileups of MCF7 CTCF, K562 RAD21 and Jurkat SMC1 ChIA-PET data showing that the majority of the ChIA-PET reads are found at the enhancer-docking site. Reads are shown as read counts per basepair. (E) The top panel depicts all putative CTCF binding motifs as blue arrows indicating the orientation of the motif. 100 vertebrate conservation from UCSC genome browser is depicted in the middle panel. The motif depicted in dark blue occurs in the most conserved region and shows the best match with consensus CTCF motif. The bottom panel shows the JASPAR score for the corresponding putative CTCF motifs in the middle panel. Below is the sequence of the motifs with the highest JASPAR score compared to the CTCF motif position weight matrix. Matching sequence is displayed in the same color as the corresponding base in the position weight matrix.

FIGS. 8A-8E. (A) Heat map of fragment lengths after genotyping PCR of wild type K562 and AK562 cells. PCR product was analyzed with a Fragment Analyzer and fragments of different lengths were quantified. (B) 4C analysis of the contacts in the MYC insulated neighborhood in wild type and 210-Δ K562 cells. Three replicate experiments for each condition shown. Black bars indicate the TAD calls, red rectangles indicate super enhancers and grey rectangles indicate typical enhancers. Top track shows H3K27Ac ChIP-seq from wild type K562 cells. (C) Perturbation of common MYC enhancer docking site reduces MYC expression and proliferation rate across cancers. Schematic representation of the experiment. Cells were transduced with one virus carrying Cas9 and two viruses each carrying one guide RNA (gRNA) under a doxycycline inducible promoter. After selection for all three components, cells were induced with doxycycline for 3 days prior to harvest and testing. (D) qPCR showing the MYC mRNA levels after deletion of the enhancer anchor site in K562, HCT-116, Jurkat and MCF7 cells. p-values were generated with a Students T-test. Error bars represent the standard deviation of the mean from three biological replicates. (E) Proliferation of parental (grey) and loop-anchor deleted (blue) K562, HCT-116, Jurkat and MCF7 cells. Error bars represent the standard deviation of the mean from six biological replicates

FIGS. 9A-9B. Generation of cells with exogenous MYC expression (MYC-cover cells). (A) qPCR measuring the mRNA levels of endogenous and exogenous MYC in parental (wild type) HCT-116 K562, MCF7 and Jurkat cells and in HCT-116 and K562 cells expressing exogenous MYC.Endogenous and exogenous MYC were detected using primers directed against the 3′ UTR of the MYC mRNA and the MYC-tdTomato junction respectively. (B) Sequencing of mutant alleles in the selected MCF7 clone with mutated enhancer docking site. The CRISPR targeted region was amplified, fragmented and sequenced to identify the composition and frequency of mutant alleles. The 6 most common mutant alleles are displayed.

FIGS. 10A-10B. The common CTCF enhancer docking site is hypomethylated in a swathe of cancer and normal cells. Percentage of methylation of CpG's at the MYC locus in (A) cander and (B) normal cells. Percent methylation of each CpG in the region is represented as a blue dot. HCT-116 CTCF ChIP-seq signal is shown in purple for reference. ChIP-seq read counts are shown in reads per million sequenced reads per basepair. Whole genome bisulfite sequencing data for a panel of healthy cells. The whole MYC gene body and the surrounding region, with the common CTCF enhancer docking site highlighted in yellow is depicted. Data from ENCODE, (Barabé et al., 2016; Ziller et al., 2013).

FIG. 11. Displays of example loci with enhancer-docking sites. ChIA-PET or HiChIP data is indicated in purple arcs with the insulated neighborhood spanning interaction in blue. CTCF ChIP-seq peaks are indicated in purple rectangles, typical enhancers are indicated in grey rectangles and super-enhancers are indicated in red rectangles. ChIA-PET data used is indicated in purple lettering. EDS=enhancer-docking site.

FIG. 12. Targeted methylation of the MYC CTCF enhancer loop anchor using dCas(-DNMT3A without the 5′ NLS in HEK293T cells. (A) Methylation at MYC promoter loop-anchor site in untreated cells, cells transfected with dCas9-DNMT3A-3L without the 5′ NLS alone or with dCas9-DNMT3A-3L without the 5′ NLS in conjunction with 4 gRNAs. (B) qPCR analysis of MYC mRNA levels and fraction of methylated CpGs for untreated, transfected and control transfected cells. dCas9-DNMT3A-3L without the 5′ NLS has reduced methlylation and a reduced effect on control transfected cells (transfected with dCas9-DNMT3A-3L without the 5′ NLS but not gRNA) as compared to dCas9-DNMT3A-3L (See FIGS. 4C and D above).

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will typically employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi) which are within the skill of the art. Non-limiting descriptions of certain of these techniques are found in the following publications: Ausubel, F., et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, N.Y., edition as of December 2008; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Freshney, R. I., “Culture of Animal Cells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, N.J., 2005. Non-limiting information regarding therapeutic agents and human diseases is found in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill/Appleton & Lange; 10th ed. (2006) or 11th edition (July 2009). Non-limiting information regarding genes and genetic disorders is found in McKusick, V. A.: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or the more recent online database: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, Md.), as of May 1, 2010, ncbi.nlm nih.gov/omim/ and in Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), at omia.angis.org.au/contact.shtml. All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.

In some aspects, the invention is directed to compositions for treating a disease or condition involving over-expression or under-expression of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51), comprising a catalytically inactive site specific nuclease fused to an effector domain having methylation activity; and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site. In one aspect, the invention is directed to compositions for treating a disease or condition involving over-expression of MYC, comprising a catalytically inactive site specific nuclease fused to an effector domain having methylation activity; and one or more guide sequences homologous or complementary to at least a portion of the MYC promoter CTCF binding site.

In some aspects, the invention is directed to compositions for treating a disease or condition involving over-expression or under-expression of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1), comprising one or more nucleic acids encoding a catalytically inactive site specific nuclease fused to an effector domain having methylation activity and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site. In some aspects, the invention is directed to compositions for treating a disease or condition involving over-expression of MYC comprising one or more nucleic acids encoding a catalytically inactive site specific nuclease fused to an effector domain having methylation activity and one or more guide sequences homologous or complementary to at least a portion of the MYC promoter CTCF binding site.

In some aspects, the composition is capable of modulating the expression (e.g., increasing or decreasing the expression) of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) by changing the degree of methylation of the promoter region CTCF binding site. In some embodiments, the composition is capable of increasing methylation of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1). In some embodiments, the composition is capable of decreasing methylation of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1). In some embodiments, the composition is capable of modulating expression of a gene having a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) by modulating the binding of CTCF to the promoter region CTCF binding site. In some embodiments, the composition is capable of decreasing binding of CTCF to the promoter region CTCF binding site. In some embodiments, the composition is capable of increasing binding of CTCF to the promoter region CTCF binding site.

In some aspects, the composition is capable of modulating the expression of MYC by changing the degree of methylation of the MYC promoter CTCF binding site. In some aspects, the composition is capable of modulating the expression of MYC by changing the degree of methylation of the MYC promoter CTCF binding site. In some embodiments, the composition is capable of increasing methylation of the MYC promoter CTCF binding site. In some embodiments, the composition is capable of decreasing methylation of the MYC promoter CTCF binding site. In some embodiments, the composition is capable of modulating MYC expression by modulating the binding of CTCF to the MYC promoter CTCF binding site. In some embodiments, the composition is capable of decreasing binding of CTCF to the MYC promoter CTCF binding site. In some embodiments, the composition is capable of increasing binding of CTCF to the MYC promoter CTCF binding site.

Without being limited to theory, it is believed based on the results shown herein that CTCF associates with the MYC promoter region and homodimerizes with a CTCF associated with a MYC enhancer region. The homodimerization forms a promoter-enhancer DNA loop, bringing enhancers into physical proximity with the MYC promoter region and increasing expression of MYC. The degree of methylation of the MYC promoter region modulates CTCF binding and modulates MYC expression.

Some aspects of the invention are directed towards a method of contacting a cell with a composition described herein and modulating promoter region CTCF binding site methylation of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51). Some aspects of the invention are directed towards a method of contacting a cell with a composition described herein and modulating expression of a gene having a promoter region CTCF binding site. Some aspects of the invention are directed towards a method of treating a patient with a disease or condition involving over-expression or under-expression of a gene having a promoter region CTCF binding site by administering a composition described herein and modulating expression of the gene. In some embodiments, the disease or condition is cancer.

In some embodiments, a CTCF binding site as used herein refers to a domain comprising one or more (e.g., 2, 3, 4, 5, 6 or more) CTCF binding motifs. In some embodiments, the CTCF binding site comprises a nucleotide sequence proximate to (e.g, within about 50 nucleotides, 75 nucleotides, 100 nucleotides, 125 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 500 nucleotides, 1000 nucleotides, 1500 nucleotides, 2000 nucleotides, 3000 nucleotides, or 5000 nucleotides) either or both ends of a CTCF binding motif. In some embodiments, the CTCF binding site comprises a domain with one or more (e.g., 2, 3, 4, 5, 6 or more) CTCF binding motifs and a nucleotide sequence proximate to (e.g, within about 50 nucleotides, 75 nucleotides, 100 nucleotides, 125 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, 500 nucleotides, 1000 nucleotides, 1500 nucleotides, 2000 nucleotides, 3000 nucleotides, or 5000 nucleotides) either or both ends of the one or more CTCF binding motifs. It is understood by a person of skill in the art that not every nucleotide in a promoter region CTCF binding site may interact with CTCF.

Some aspects of the invention are directed towards a method of contacting a cell with a composition described herein and modulating MYC promoter CTCF binding site methylation. Some aspects of the invention are directed towards a method of contacting a cell with a composition described herein and modulating MYC expression. Some aspects of the invention are directed towards a method of treating a patient with a disease or condition involving MYC over-expression or under-expression by administering a composition described herein and modulating MYC expression. In some embodiments, the disease or condition is cancer and administration of the composition reduces MYC expression.

The MYC promoter CTCF binding site as used herein refers to a domain of the MYC promoter comprising multiple (e.g., 2, 3, 4, 5, 6 or more) CTCF binding motifs. It is understood by a person of skill in the art that not every nucleotide in the MYC promoter CTCF binding site may interact with CTCF.

The terms “disease,” “disorder” or “condition” are used interchangeably and may refer to any alteration from a state of health and/or normal functioning of an organism, e.g., an abnormality of the body or mind that causes pain, discomfort, dysfunction, distress, degeneration, or death to the individual afflicted. Diseases include any disease known to those of ordinary skill in the art. In some embodiments a disease is a chronic disease, e.g., it typically lasts or has lasted for at least 3-6 months, or more, e.g., 1, 2, 3, 5, 10 or more years, or indefinitely. Disease may have a characteristic set of symptoms and/or signs that occur commonly in individuals suffering from the disease. Diseases and methods of diagnosis and treatment thereof are described in standard medical textbooks such as Longo, D., et al. (eds.), Harrison's Principles of Internal Medicine, 18th Edition; McGraw-Hill Professional, 2011 and/or Goldman's Cecil Medicine, Saunders; 24 edition (Aug. 5, 2011). In certain embodiments a disease is a multigenic disorder (also referred to as complex, multifactorial, or polygenic disorder). Such diseases may be associated with the effects of multiple genes, sometimes in combination with environmental factors (e.g., exposure to particular physical or chemical agents or biological agents such as viruses, lifestyle factors such as diet, smoking, etc.). A multigenic disorder may be any disease for which it is known or suspected that multiple genes (e.g., particular alleles of such genes, particular polymorphisms in such genes) may contribute to risk of developing the disease and/or may contribute to the way the disease manifests (e.g., its severity, age of onset, rate of progression, etc.) In some embodiments a multigenic disease is a disease that has a genetic component as shown by familial aggregation (occurs more commonly in certain families than in the general population) but does not follow Mendelian laws of inheritance, e.g., the disease does not clearly follow a dominant, recessive, X-linked, or Y-linked inheritance pattern. In some embodiments a multigenic disease is one that is not typically controlled by variants of large effect in a single gene (as is the case with Mendelian disorders). In some embodiments a multigenic disease may occur in familial form and sporadically. Examples include, e.g., Parkinson's disease, Alzheimer's disease, and various types of cancer. Examples of multigenic diseases include many common diseases such as hypertension, diabetes mellitus (e.g., type II diabetes mellitus), cardiovascular disease, cancer, and stroke (ischemic, hemorrhagic). In some embodiments a disease, e.g., a multigenic disease is a psychiatric, neurological, neurodevelopmental disease, neurodegenerative disease, cardiovascular disease, autoimmune disease, cancer, metabolic disease, or respiratory disease. In some embodiments the disease or condition involves overexpression of MYC. In some embodiments the disease or condition involves aberrant expression of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1.

In some embodiments, the disease or condition involving over-expression of MYC, or aberrant expression of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1, is cancer which term is generally used interchangeably to refer to a disease characterized by one or more tumors, e.g., one or more malignant or potentially malignant tumors. The term “tumor” as used herein encompasses abnormal growths comprising aberrantly proliferating cells. As known in the art, tumors are typically characterized by excessive cell proliferation that is not appropriately regulated (e.g., that does not respond normally to physiological influences and signals that would ordinarily constrain proliferation) and may exhibit one or more of the following properties: dysplasia (e.g., lack of normal cell differentiation, resulting in an increased number or proportion of immature cells); anaplasia (e.g., greater loss of differentiation, more loss of structural organization, cellular pleomorphism, abnormalities such as large, hyperchromatic nuclei, high nuclear to cytoplasmic ratio, atypical mitoses, etc.); invasion of adjacent tissues (e.g., breaching a basement membrane); and/or metastasis. Malignant tumors have a tendency for sustained growth and an ability to spread, e.g., to invade locally and/or metastasize regionally and/or to distant locations, whereas benign tumors often remain localized at the site of origin and are often self-limiting in terms of growth. The term “tumor” includes malignant solid tumors, e.g., carcinomas (cancers arising from epithelial cells), sarcomas (cancers arising from cells of mesenchymal origin), and malignant growths in which there may be no detectable solid tumor mass (e.g., certain hematologic malignancies). Cancer includes, but is not limited to: breast cancer; biliary tract cancer; bladder cancer; brain cancer (e.g., glioblastomas, medulloblastomas); cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms including acute lymphocytic leukemia and acute myelogenous leukemia; T-cell acute lymphoblastic leukemia/lymphoma; hairy cell leukemia; chronic lymphocytic leukemia, chronic myelogenous leukemia, multiple myeloma; adult T-cell leukemia/lymphoma; intraepithelial neoplasms including Bowen's disease and Paget's disease; liver cancer; lung cancer; lymphomas including Hodgkin's disease and lymphocytic lymphomas; neuroblastoma; melanoma, oral cancer including squamous cell carcinoma; ovarian cancer including ovarian cancer arising from epithelial cells, stromal cells, germ cells and mesenchymal cells; neuroblastoma, pancreatic cancer; prostate cancer; rectal cancer; sarcomas including angiosarcoma, gastrointestinal stromal tumors, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; renal cancer including renal cell carcinoma and Wilms tumor; skin cancer including basal cell carcinoma and squamous cell cancer; testicular cancer including germinal tumors such as seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumors, and germ cell tumors; thyroid cancer including thyroid adenocarcinoma and medullary carcinoma. It will be appreciated that a variety of different tumor types can arise in certain organs, which may differ with regard to, e.g., clinical and/or pathological features and/or molecular markers. Tumors arising in a variety of different organs are discussed, e.g., the WHO Classification of Tumours series, 4^(th) ed, or 3^(rd) ed (Pathology and Genetics of Tumours series), by the International Agency for Research on Cancer (IARC), WHO Press, Geneva, Switzerland, all volumes of which are incorporated herein by reference. In some embodiments, the cancer is lung cancer, breast cancer, cervical cancer, colon cancer, gastric cancer, kidney cancer, leukemia, liver cancer, lymphoma, (e.g., a Non-Hodgkin lymphoma, e.g., diffuse large B-cell lymphoma, Burkitts lymphoma) ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcoma, skin cancer, testicular cancer, or uterine cancer. The type of cancer is not limited as long as over-expression of MYC, or aberrant expression of aberrant expression of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1, is exhibited. In some embodiments, the cancer is colorectal cancer, leukemia (e.g., acute T-cell leukemia, Chronic Myeloid Leukemia), or breast cancer. In some embodiments the cancer is neuroblastoma and administering the composition increases methylation of the CTCF binding site within the N-MYC promoter, thereby decreasing expression of N-MYC. In some embodiments the cancer is lung cancer and administering the composition increases methylation of the CTCF binding site within the L-MYC promoter, thereby decreasing expression of L-MYC.

In some embodiments, a method of treating a subject in need of treatment for cancer comprises measuring expression and/or activity of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1 in a cancer (e.g., in a sample obtained from a cancer (e.g., a biopsy sample, circulating cancer cells, etc.)); determining that the cancer comprises cells with aberrant expression of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1 relative to a reference value; and administering a composition disclosed herein that modulates expression of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1 to the subject. Measuring expression may comprise measuring mRNA or protein. Measuring activity may comprise measuring expression of one or more target genes of TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1. In some embodiments a reference value may be a value for a normal, non-cancer cell, e.g., of the same cell type as the cancer cell. In some aspects, a method of treating cancer may further comprise administering a second ant-cancer agent (e.g., a conventional chemotherapy agent, a molecularly targeted therapy, a cancer immunotherapy agent, radiotherapy, or a combination thereof).

In some embodiments a method of treating a subject in need of treatment for cancer comprises measuring expression and/or activity of MYC in a cancer (e.g., in a sample obtained from a cancer (e.g., a biopsy sample, circulating cancer cells, etc.)); determining that the cancer comprises cells that over-express MYC relative to a reference value; and administering a composition disclosed herein that reduces MYC expression to the subject. Measuring expression may comprise measuring MYC mRNA or protein. Measuring MYC activity may comprise measuring expression of one or more MYC target genes. In some embodiments a reference value may be a value for a normal, non-cancer cell, e.g., of the same cell type as the cancer cell. In some embodiments a method comprises determining that the cancer comprises cells that harbor three or more copies of MYC (MYC amplification) or a MYC translocation; and administering a composition disclosed herein that reduces MYC expression to the subject. In some aspects, a method of treating cancer may further comprise administering a second ant-cancer agent (e.g., a conventional chemotherapy agent, a molecularly targeted therapy, a cancer immunotherapy agent, radiotherapy, or a combination thereof).

In some embodiments, the disease or condition involving over-expression of the MYC gene is a proliferative disease such as restenosis or polycystic kidney disease.

By “MYC” as used herein refers to nucleic acid sequences encoding any MYC protein, peptide, or polypeptide having MYC activity. The term “MYC” is also meant to include other MYC encoding sequence, such as MYC isoforms (e.g., N-MYC, L-MYC, etc.), mutant MYC genes, splice variants of MYC genes, and MYC gene polymorphisms. In some embodiments, MYC is NCBI Gene ID 4609

In some embodiments, the MYC promoter CTCF binding site is located 2 kb upstream of the major transcript start site (E. M. Klenova et al., ref (32), incorporated by reference in its entirety). In some embodiments, the MYC promoter CTCF binding site is located at Chr8:128746041-128746751 (Genome build GR37/HG19). In some embodiments, the MYC is N-MYC. In some embodiments, the N-MYC promoter CTCF binding site is located at Chr2:16079556-16080469 (Genome build GR37/HG19). In some embodiments, the MYC is L-MYC. In some embodiments, the L-MYC promoter binding site is located at chr1:40367702-40368974 (Genome build GR37/HG19).

As used herein, the terms “site specific nuclease” and “a targetable nuclease” are used interchangeably. Site specific nucleases and targetable nucleases are known in the art. See U.S. Pat. Pub. Nos. 20140068797, 20140186919, 20140170753 and WO/2014/172470, incorporated herein by reference in their entireties. In some embodiments, a site specific nuclease is a targetable nuclease. In some embodiments, a targetable nuclease is a site specific nuclease. In some embodiments, the site-specific nuclease is a Cas protein. In some embodiments, the site-specific nuclease is catalytically inactive. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas protein. A variety of CRISPR associated (Cas) genes or proteins which are known in the art can be used in the compositions and methods of the invention and the choice of Cas protein will depend upon the particular situation (e.g., www.ncbi.nlm.nih.gov/gene/?term=cas9). Specific examples of Cas proteins include Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 and Cas10. In a particular aspect, the Cas nucleic acid or protein used in the compositions is Cas9. In some embodiments a Cas protein, e.g., a Cas9 protein, may be from any of a variety of prokaryotic species. In some embodiments a particular Cas protein, e.g., a particular Cas9 protein, may be selected to recognize a particular protospacer-adjacent motif (PAM) sequence. In certain embodiments a Cas protein, e.g., a Cas9 protein, may be obtained from a bacteria or archaea or synthesized using known methods. In certain embodiments, a Cas protein may be from a gram positive bacteria or a gram negative bacteria. In certain embodiments, a Cas protein may be from a Streptococcus, (e.g., a S. pyogenes, a S. thermophilus) a Cryptococcus, a Corynebacterium, a Haemophilus, a Eubacterium, a Pasteurella, a Prevotella, a Veillonella, or a Marinobacter. In some embodiments nucleic acids encoding two or more different Cas proteins, or two or more Cas proteins, may be present in the composition, e.g., to allow for recognition and modification of sites comprising the same, similar or different PAM motifs.

In some embodiments, the Cas protein is Cpf1 protein or a functional portion thereof. In some embodiments, the Cas protein is Cpf1 from any bacterial species or functional portion thereof. In certain embodiments, a Cpf1 protein is a Francisella novicida U112 protein or a functional portion thereof, a Acidaminococcus sp. BV3L6 protein or a functional portion thereof, or a Lachnospiraceae bacterium ND2006 protein or a functional portion thereof. Cpf1 protein is a member of the type V CRISPR systems. Cpf1 protein is a polypeptide comprising about 1300 amino acids. Cpf1 contains a RuvC-like endonuclease domain. Catalytically inactive Cpf1 is known in the art. See US Pat. Pub. No. 20160208243, incorporated by reference in its entirety.

In some embodiments, the Cas protein is a variant polypeptide with at least about 50%, 60%. 70%, 80%, 90%, 95%, or 99% sequence identity to a naturally occurring Cas protein.

In some embodiments a Cas9 nickase may be generated by inactivating one or more of the Cas9 nuclease domains. In some embodiments, an amino acid substitution at residue 10 in the RuvC I domain of Cas9 converts the nuclease into a DNA nickase. For example, the aspartate at amino acid residue 10 can be substituted for alanine (Cong et al, Science, 339:819-823). Other amino acids mutations that create a catalytically inactive Cas9 protein include mutating at residue 10 and/or residue 840. Mutations at both residue 10 and residue 840 can create a catalytically inactive Cas9 protein, sometimes referred herein as dCas9. In some embodiments, dCas9 is a D10A and a H840A Cas9 mutant that is catalytically inactive.

In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas9 protein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cpf1 protein.

As used herein an “effector domain” is a molecule (e.g., protein) that modulates the expression and/or activation of a genomic sequence (e.g., gene). The effector domain may have methylation activity (e.g., DNA methylation activity). In some aspects, the effector domain targets one or both alleles of a gene. The effector domain can be introduced as a nucleic acid sequence and/or as a protein. In some aspects, the effector domain can be a constitutive or an inducible effector domain. In some aspects, a Cas (e.g., dCas) nucleic acid sequence or variant thereof and an effector domain nucleic acid sequence are introduced into the cell as a chimeric sequence. In some aspects, the effector domain is fused to a molecule that associates with (e.g., binds to) Cas protein (e.g., the effector molecule is fused to an antibody or antigen binding fragment thereof that binds to Cas protein). In some aspects, a Cas (e.g., dCas) protein or variant thereof and an effector domain are fused or tethered creating a chimeric protein and are introduced into the cell as the chimeric protein. In some aspects, the Cas (e.g., dCas) protein and effector domain bind as a protein-protein interaction. In some aspects, the Cas (e.g., dCas) protein and effector domain are covalently linked. In some aspects, the effector domain associates non-covalently with the Cas (e.g., dCas) protein. In some aspects, a Cas (e.g., dCas) nucleic acid sequence and an effector domain nucleic acid sequence are introduced as separate sequences and/or proteins. In some aspects, the Cas (e.g., dCas) protein and effector domain are not fused or tethered.

As shown herein, fusions of a catalytically inactive (D10A; H840A) Cas9 protein (dCas9) tethered with all or a portion of (e.g., biologically active portion of) an (one or more) effector domain create chimeric proteins that can be guided to specific DNA sites by one or more RNA sequences (sgRNA) to modulate activity and/or expression of MYC. In specific aspects, fusions of a dCas9 tethered with all or a portion of an effector domain create chimeric proteins that can be guided to specific DNA sites by one or more RNA sequences to modulate or modify methylation of MYC. As used herein, a “biologically active portion of an effector domain” is a portion that maintains the function (e.g. completely, partially, minimally) of an effector domain (e.g., a “minimal” or “core” domain). The fusion of the Cas9 (e.g., dCas9) with all or a portion of one or more effector domains created a chimeric protein.

Examples of effector domains include a transcription(al) activating domain, a coactivator domain, a transcription factor, a transcriptional pause release factor domain, a negative regulator of transcriptional elongation domain, a transcriptional repressor domain, a chromatin organizer domain, a remodeler domain, a histone modifier domain, a DNA modification domain, a RNA binding domain, a protein interaction input device domain (Grunberg and Serrano, Nucleic Acids Research, 38 (8): 2663-2675 (2010), and a protein interaction output device domain (Grunberg and Serrano, Nucleic Acids Research, 38 (8): 2663-2675 (2010). As used herein a “protein interaction input device” and a “protein interaction output device” refers to a protein-protein interaction (PPI). In some aspect, binding partners are targeted to different sites in the genome using the catalytically inactive Cas protein. The binding partners interact, thereby bringing the targeted loci into proximity.

In some aspects, the effector domain is a DNA modifier. Specific examples of DNA modifiers include 5hmc conversion from 5mC such as Tet1 (Tet1CD); DNA demethylation by Tet1, ACID A, MBD4, Apobec1, Apobec2, Apobec3, Tdg, Gadd45a, Gadd45b, ROS1; DNA methylation by Dnmtl, DNMT3A, Dnmt3b, CpG Methyltransferase M.SssI, and/or M.EcoHK31I. In specific aspects, an effector domain is DNMT3A. In some aspects, the effector domain is the C-terminal domain of DNMT3A (i.e., DNMT3A-C). In some aspects, the DNMT3A-C effector domain is complexed with the C-terminal portion of DNMT3L (DNMT3L-C). In some aspects, a chimeric protein comprising DNMT3A-C and DNMT3L-C (sometimes referred to herein as DNMT3A-3L) is used for the effector domain. In some aspects, DNMT3A-3L is a single chain fusion protein as provided in Siddique, et al. (2013) incorporated herein by reference in its entirety. In some embodiments, the effector domain is DNMT3A-3L without the 5′ NLS. In some embodiments, dCas9 is fused to DNMT3A-3L or DNMT3A-3L without the 5′ NLS.

DNA methylation is established by two de novo DNA methyltransferases (DNMT3A/B), and is maintained by DNMT1 (Smith and Meissner, (2013). DNA methylation: roles in mammalian development. Nature reviews Genetics 14, 204-220). Gene activation during development is associated with demethylation of promoter and enhancer sequences. In addition, demethylation can be achieved through oxidation of the methyl group by TET (ten-eleven translocation) dioxygenases to form 5-hydroxymethylcytosine (5-hmC), and then restoration into unmodified cytosines by either DNA replication-dependent dilution or DNA glycosylase-initiated base excision repair (BER), a process termed as active demethylation and proposed to operate during specific developmental stages such as preimplantation embryos or in post-mitotic neurons.

In one aspect of the invention, fusion of the dCas9 to an effector domain can be to that of a single copy or multiple/tandem copies of full-length or partial-length effectors. Other fusions can be with split (functionally complementary) versions of the effector domains. In some embodiments, the effector domain can include full-length or partial-length effectors from more than one effector (e.g., DNMT3A and DNMT3L). Effector domains for use in the methods include any one of the following classes of proteins: proteins that mediate drug inducible looping of DNA and/or contacts of genomic loci, proteins that aid in the three-dimensional proximity of genomic loci bound by dCas9 with different sgRNA.

Other examples of effector domains are described in PCT Application No. PCT/US2014/034387 and U.S. application Ser. No. 14/785,031, which are incorporated herein by reference in their entirety.

In some embodiments, the catalytically inactive site specific nuclease fused to an effector domain having DNA methylation activity is dCas9-DMNT3A-3L or dCas9-DNMT3A-3L without the 5′ NLS.

In some aspects the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a nucleic acid sequence that encodes a fusion protein (chimeric protein) comprising all or a portion of a Cas (e.g., dCas) protein fused to all or a portion of one or more effector domains. In some aspects, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a fusion protein comprising all or a portion of a Cas (e.g., dCas) protein fused to all or a portion of one or more effector domains. In some aspects all or a portion of the Cas (e.g., dCas) protein targets but does not cleave a nucleic acid sequence. In some aspects, the Cas (e.g., dCas) protein can be fused to the N-terminus or C-terminus of one or more effector domains. In some aspects, the portion of the effector domain modulates the methylation of the genomic sequence (e.g., demethylates or methylates the genomic sequence). In some aspects, the effector domain comprises the c-terminal portions of DNMT3A and DNMT3L. In some aspects, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a fusion protein comprising dCas9-DNMT3A-3L or dCas9-DNMT3A-3L without the 5′ NLS.

In some aspects, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a catalytically inactive nuclease (e.g., dCas9), an effector domain (e.g., DNMT3a, DMNT3A-C, DMNT3A-3L, DNMT3A-3L without the 5′ NLS) and one or more guide sequences. In some aspects, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) nucleic acids encoding a catalytically inactive nuclease (e.g., dCas9), an effector domain (e.g., DNMT3a, DMNT3A-C, DMNT3A-3L, DNMT3A-3L without the 5′ NLS) and one or more guide sequences.

In some aspects, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a mixture of nucleic acids and polypeptides. In some embodiments, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a catalytically inactive nuclease (e.g., dCas9), an effector domain (e.g., DNMT3a, DMNT3A-C, DMNT3A-3L, DNMT3A-3L without the 5′ NLS) and one or more nucleic acids encoding one or more guide sequences. In some embodiments, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a catalytically inactive nuclease (e.g., dCas9) and effector (e.g., DNMT3a, DMNT3A-C, DMNT3A-3L, DNMT3A-3L without the 5′ NLS) fusion protein and one or more nucleic acids encoding one or more guide sequences. In some embodiments, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) one or more nucleic acids encoding a catalytically inactive nuclease (e.g., dCas9) and an effector domain (e.g., DNMT3a, DMNT3A-C, DMNT3A-3L, DNMT3A-3L without the 5′ NLS) and one or more nucleic acids encoding one or more guide sequences. In some embodiments, the invention is directed to (e.g., a composition comprising, consisting essentially of, consisting of) a catalytically inactive nuclease (e.g., dCas9) and effector (e.g., DNMT3a, DMNT3A-C, DMNT3A-3L, DNMT3A-3L without the 5′ NLS) fusion protein and one or more nucleic acids encoding one or more guide sequences. Every combination of encoding nucleic acids (e.g., encoding a catalytically inactive site specific nuclease, effector, catalytically inactive nuclease-effector fusion protein, and/or guide sequence) with or without non-encoded components (e.g., a catalytically inactive site specific nuclease, effector, catalytically inactive nuclease-effector fusion protein, and/or guide sequence) having the capability to modulate MYC expression are contemplated herein.

In some aspects, the nucleic acid sequence encoding the fusion protein and/or the one or more guide sequences are isolated. An “isolated,” “substantially pure,” or “substantially pure and isolated” nucleic acid sequence, as used herein, is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA or cDNA library). For example, an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. An “isolated,” “substantially pure,” or “substantially pure and isolated” protein (e.g., chimeric protein; fusion protein), as used herein, is one that is separated from or substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system, or reagent mix. In other circumstances, the material may be purified to essential homogeneity, for example, as determined by agarose gel electrophoresis or column chromatography such as HPLC. Preferably, an isolated nucleic acid molecule comprises at least about 50%, 80%, 90%, 95%, 98% or 99% (on a molar basis) of all macromolecular species present.

In one aspect, fusion of catalytically inactive site specific nuclease (e.g, a catalytically inactive Cas protein) with all or a portion of one or more effector domains comprise one or more linkers. As used herein, a “linker” is something that connects or fuses two or more moieties (e.g see Hermanson, Bioconjugate Techniques, 2^(nd) Edition, which is hereby incorporated by reference in its entirety). As will be appreciated by one of ordinary skill in the art, a variety of linkers can be used. In one aspect, a linker comprises one or more amino acids. In some aspects, a linker comprises two or more amino acids. In one aspect, a linker comprises the amino acid sequence GS. In some aspects, fusion of Cas9 (e.g., dCas9) with two or more effector domains comprises one or more interspersed linkers (e.g., GS linkers) between the domains. In some aspects, one or more nuclear localization sequences may be located between the catalytically inactive nuclease (e.g., dCas9) and the effector domain. For example, a fusion protein may include dCas9-NLS-DNMT3A or dCas9-NLS-DNMT3A-3L. In some embodiments, the one or more nuclear localization sequences may be located anywhere in the fusion protein. In some embodiments, the fusion protein does not comprise a NLS, or does not comprise an NLS located between the catalytically inactive nuclease (e.g., dCas9) and the effector domain.

In some aspects, one or more guide sequences include sequences that recognize DNA in a site-specific manner. For example, guide sequences can include guide ribonucleic acid (RNA) sequences utilized by a CRISPR system or sequences within a TALEN or zinc finger system that recognize DNA in a site-specific manner. In some embodiments, the guide sequences comprise a portion that is complementary or homologous to a portion of each of the one or more genomic sequences and comprise a binding site for the catalytically inactive site specific nuclease. In some embodiments, one or more guide sequences do not comprise a binding site for the catalytically inactive site specific nuclease. In some embodiments, one or more guide sequences comprise a moity that blocks (e.g., sterically blocks) CTCF binding when the one or more guide sequences is bound to genomic sequences. In some embodiments, the guide sequence is referred to as guide RNA (gRNA) or single guide RNA (sgRNA).

In some aspects, a single guide sequence can be complementary or homologous to one or more (e.g., all) of the genomic sequences that are being modulated or modified. In one aspect, a single guide is complementary or homologous to a single target genomic sequence. In a particular aspect in which two or more target genomic sequences are to be modulated or modified, multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) guide sequences are introduced wherein each guide sequence is complementary or homologous to (specific for) one target genomic sequence. In some aspects, two or more, three or more, four or more, five or more, or six or more guide sequences are complementary or homologous to (specific for) different parts of the same target sequence. In one aspect, two or more guide sequences bind to different sequences of the same region of DNA. In some aspects, a single guide sequence is complementary or homologous to at least two target or more (e.g., all) of the genomic sequences. It will also be apparent to those of skill in the art that the portion of the guide sequence that is complementary or homologous to one or more of the genomic sequences and the portion of the guide sequence that binds to the catalytically inactive site specific nuclease can be introduced as a single sequence or as 2 (or more) separate sequences into a cell. In some embodiments the sequence that binds to the catalytically inactive site specific nuclease comprises a stem-loop.

In some embodiments, one or more guide sequences comprise a sequence homologous or complementary to a portion of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1). In some embodiments, one or more guide sequences comprise a sequence homologous or complementary to a nucleotide sequence of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) and/or a sequence within 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nt of the promoter region CTCF binding site. The portion of the guide sequence homologous or complementary to the promoter region CTCF binding site or adjacent sequence (e.g., within 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nt of the promoter region CTCF binding site) can comprise 15 nt or more, 18 nt or more, or 20 nt or more.

In some embodiments, one or more guide sequences comprise a sequence homologous or complementary to a portion of the MYC promoter CTCF binding site. In some embodiments, one or more guide sequences comprise a sequence homologous or complementary to a nucleotide sequence of the MYC promoter CTCF binding site and/or a sequence within 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nt of the MYC promoter CTCF binding site. The portion of the guide sequence homologous or complementary to the MYC promoter CTCF binding site or adjacent sequence (e.g., within 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nt of the MYC promoter CTCF binding site) can comprise 15 nt or more, 18 nt or more, or 20 nt or more.

In some embodiments, guide sequence used to modify gene expression (e.g., MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1 gene expression) is a naturally occurring RNA sequence, a modified RNA sequence (e.g., a RNA sequence comprising one or more modified bases), a synthetic RNA sequence, or a combination thereof. As used herein a “modified RNA” is an RNA comprising one or more modifications (e.g., RNA comprising one or more non-standard and/or non-naturally occurring bases and/or modifications to the backbone, internucleoside linkage(s) and/or sugar). Methods of modifying bases of RNA are well known in the art. Examples of such modified bases include those contained in the nucleosides 5-methylcytidine (5mC), pseudouridine (Ψ), 5-methyluridine, 2′O-methyluridine, 2-thiouridine, N-6 methyladenosine, hypoxanthine, dihydrouridine (D), inosine (I), and 7-methylguanosine (m7G). It should be noted that any number of bases, sugars, or backbone linkages in a RNA sequence can be modified in various embodiments. It should further be understood that combinations of different modifications may be used. In some embodiments an RNA comprises one or more modifications selected from: phosphorothioate, 2′-OMe, 2′-F, 2′-constrained ethyl (2′-cEt), 2′-OMe 3′ phosphorothioate (MS), and 2′-OMe 3-thioPACE (MSP) modifications. In some embodiments a modification may stabilize the RNA and/or increase its binding affinity to a complementary sequence.

In some embodiments, the one or more guide sequences comprise at least one locked nucleic acid (LNA) unit, such as 1, 2, 3, 4, 5, 6, 7, or 8 LNA units, such as from about 3-7 or 4-8 LNA units, or 3, 4, 5, 6 or 7 LNA units. In some embodiments, all the nucleotides of the one or more guide sequences are LNA. In some embodiments, the one or more guide sequences may comprise both beta-D-oxy-LNA, and one or more of the following LNA units: thio-LNA, amino-LNA, oxy-LNA, and/or ENA in either the beta-D or alpha-L configurations or combinations thereof. In some embodiments all LNA cytosine units are 5′methyl-cytosine.

In some aspects, the RNA sequence is a morpholino. Morpholinos are typically synthetic molecules, of about 25 bases in length and bind to complementary sequences of RNA by standard nucleic acid base-pairing. Morpholinos have standard nucleic acid bases, but those bases are bound to morpholine rings instead of deoxyribose rings and are linked through phosphorodiamidate groups instead of phosphates. Morpholinos do not degrade their target RNA molecules, unlike many antisense structural types (e.g., phosphorothioates, siRNA). Instead, morpholinos act by steric blocking and bind to a target sequence within a RNA and block molecules that might otherwise interact with the RNA.

In some embodiments, an RNA sequence can vary in length from about 8 base pairs (bp) to about 200 bp. In some embodiments, each of the one or more guide sequences can be about 9 to about 190 bp; about 10 to about 150 bp; about 15 to about 120 bp; about 20 to about 100 bp; about 30 to about 90 bp; about 40 to about 80 bp; about 50 to about 70 bp in length.

Chemical modifications and methods of synthesizing guide RNAs (guide sequences) are known in the art. See WO/2016/164356, herein incorporated by reference in its entirety.

The portion of each genomic sequence (e.g., MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1 promoter region CTCT binding sequence) to which each guide sequence is complementary or homologous to can also vary in size. In particular aspects, the portion of each genomic sequence to which the guide sequence is complementary or homologous to can be about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38 39, 40, 41, 42, 43, 44, 45, 46 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 81, 82, 83, 84, 85, 86, 87 88, 89, 90, 81, 92, 93, 94, 95, 96, 97, 98, or 100 nucleotides (contiguous nucleotides) in length. In some embodiments, each guide sequence can be at least about 70%, 75%, 80%, 85%, 90%, 95%, 100%, etc. identical, complementary or similar to the portion of each genomic sequence. In some embodiments, each guide sequence is completely or partially identical, complementary or similar to each genomic sequence. For example, each guide sequence can differ from perfect complementarity or homology to the portion of the genomic sequence by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. nucleotides. In some embodiments, one or more guide sequences are perfectly complementary or homologous (100%) across at least about 10 to about 25 (e.g., about 20) nucleotides of the genomic sequence.

As will be apparent to those of ordinary skill in the art, the one or more RNA sequences can further comprise one or more expression control elements. For example, in some embodiments the RNA sequences comprises a promoter, suitable to direct expression in cells, wherein the portion of the RNA sequence is operably linked to the expression control element(s). The promoter can be a viral promoter (e.g., a CMV promoter) or a mammalian promoter (e.g., a PGK promoter). The RNA sequence can comprise other genetic elements, e.g., to enhance expression or stability of a transcript. In some embodiments the additional coding region encodes a selectable marker (e.g., a reporter gene such as green fluorescent protein (GFP)).

As described herein, the one or more guide sequences also comprise a (one or more) binding site for a (one or more) catalytically inactive site specific nuclease. The catalytically inactive site specific nuclease may be a catalytically inactive CRISPR associated (Cas) protein. In a particular aspect, upon hybridization of the one or more guide sequences to the one or more genomic sequences, the catalytically inactive site specific nuclease binds to the one or more guide sequences.

In some aspects, the guide sequences are ribonucleic acid guide sequences. In some aspects, each guide sequence is from about 10 base pairs to about 150 base pairs in length. In some aspects, the composition comprises at least two guide sequences. In some aspects, the compositions and methods disclosed herein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more guide sequences. In some embodiments, the compositions and methods disclosed herein can comprise 1, 2 or 5 guide sequences.

In some aspects, the one or more guide sequences comprise a sequence homologous to a sequence selected from the group consisting of SEQ ID NOS. 1-8.

SEQ ID NO: 1 GCCTGGATGTCAACGAGGGC SEQ ID NO: 2 GCGGGTGCTGCCCAGAGAGG SEQ ID NO: 3 GCAAAATCCAGCATAGCGAT SEQ ID NO: 4 CTATTCAACCGCATAAGAGA SEQ ID NO: 5 CGCTGAGCTGCAAACTCAAC SEQ ID NO: 6 ACCGCCTGTCCTTCCCCCGC SEQ ID NO: 7 TTGGTTGCTCCCCGCGTTTG SEQ ID NO: 8 ATGATCTCTGCTGCCAGTAG

There are various ways that a polypeptide comprising a catalytically inactive site specific nuclease fused to an effector domain having methylation activity can be delivered to a cell or subject, e.g., by administering a nucleic acid that encodes the polypeptide, which nucleic acid may be, e.g., a viral vector or may be a translatable nucleic acid (e.g, synthetic modified mRNA. In some embodiments a nucleic acid sequence encoding a polypeptide is codon optimized for expression in mammalian cells, e.g., human cells. Examples of modified mRNA are described in Warren et al. (Cell Stem Cell 7(5):618-30, 2010, Mandal P K, Rossi D J. Nat Protoc. 2013 8(3):568-82, US Pat. Pub. No. 20120046346 and/or PCT/US2011/032679 (WO/2011/130624). Additional examples are found in numerous PCT and US applications and issued patents to Moderna Therapeutics, e.g., PCT/US2011/046861; PCT/US2011/054636, PCT/US2011/054617, U.S. Ser. No. 14/390,100 (and additional patents and patent applications mentioned in these.) In some embodiments the guide sequence can be delivered as a nucleic acid that encodes the guide sequence. In some embodiments a nucleic acid comprises a first portion that encodes a polypeptide comprising a catalytically inactive site-specific nuclease fused to an effector domain and a second portion that encodes a guide RNA. One of ordinary skill in the art will appreciate that a nucleic acid that encodes a polypeptide or RNA may be operably linked to a promoter capable of directing expression in a cell or subject, e.g., a mammalian cell or subject.

Administration may be by any route (e.g., oral, intravenous, intraperitoneal, gavage, topical, transdermal, intramuscular, enteral, subcutaneous), may be systemic or local, may include any dose (e.g., from about 0.01 mg/kg to about 500 mg/kg), may involve a single dose or multiple doses. In some embodiments administration may be performed by direct administration to a tissue or organ (e.g., skin, heart, liver, lung, kidney, brain, eye, muscle, bone, nerve) or tumor. The nucleic acid(s) or protein(s) may be physically associated with, e.g., encapsulated, e.g., in lipid-containing particles, e.g., solid lipid nanoparticles, liposomes, polymeric particles (e.g., PLGA particles). In some embodiments one or more nucleic acids may be administered using a vector (e.g., a viral vector such as an adenoviral vector, lentiviral vector, or adeno-associated virus vector). In some embodiments one or more nucleic acids, proteins, and/or vectors may be combined with a pharmaceutically acceptable carrier to produce a pharmaceutical composition, which may be administered to a subject.

In some embodiments a nucleic acid, polypeptide, or particle may be targeted to cells of a particular type, e.g., cancer cells of a particular type or expressing a particular cell surface marker. For example, a nucleic acid, protein, or a particle comprising a nucleic acid or vector may comprise or be conjugated to a targeting moiety that binds to a marker expressed at the surface of a target cell (e.g., binds to a tumor antigen or a receptor expressed by the target cell). A targeting moiety may comprise, e.g., an antibody or antigen-binding portion thereof, an engineered protein capable of specific binding, a nucleic acid aptamer, a ligand, etc.

In some embodiments, nucleic acids encoding one or more components (e.g., catalytically inactive site specific nuclease, effector domain, catalytically inactive site specific nuclease-effector domain fusion protein, one or more guide sequences) are delivered by one or more viral vectors e.g., a retroviral vector such as a lentiviral vector or gamma retroviral vector, or an adenoviral or AAV vector. In some embodiments, the nucleic acids encoding a catalytically inactive site specific nuclease, effector domain, and/or catalytically inactive site specific nuclease-effector domain fusion protein are codon-optimized for expression in a subject (e.g., human).

In some aspects, the invention is directed towards a composition that inhibits binding of CTCF to a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1). In some embodiments, the composition comprises a small molecule or a nucleic acid derivative. In some embodiments, the composition binds to CTCF. In some embodiments, the composition binds to a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1).

Also disclosed herein are methods for methylating a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) in a cell comprising introducing into the cell a catalytically inactive site specific nuclease fused to an effector domain having methylation activity; and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site, thereby methylating the promoter region CTCF binding site.

In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas protein as described herein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas9 protein as described herein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cpf1 protein as described herein. In some embodiments, the effector domain having methylation activity is DMNT3A-3L or DMNT3A-3L without a 5′ NLS as described herein. In some embodiments, the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DMNT3A-3L or dCas9-DMNT3A-3L without a 5′ NLS as described herein. In some aspects, expression of a gene with a promoter region CTCF binding site is modulated. In some aspects, expression of a gene with a promoter region CTCF binding site is decreased. In some aspects, expression of a gene with a promoter region CTCF binding site is increased.

In some aspects, the invention is directed towards a composition that inhibits binding of CTCF to the MYC promoter CTCF binding site. In some embodiments, the composition comprises a small molecule or a nucleic acid derivative. In some embodiments, the composition binds to CTCF. In some embodiments, the composition binds to the MYC promoter CTCF binding site.

Also disclosed herein are methods for methylating a MYC promoter CTCF binding site in a cell comprising introducing into the cell a catalytically inactive site specific nuclease fused to an effector domain having methylation activity; and one or more guide sequences homologous or complementary to at least a portion of the MYC promoter CTCF binding site, thereby methylating the MYC promoter CTCF binding site.

In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas protein as described herein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas9 protein as described herein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cpf1 protein as described herein. In some embodiments, the effector domain having methylation activity is DMNT3A-3L as described herein. In some embodiments, the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DMNT3A-3L as described herein. In some aspects, MYC expression is modulated. In some aspects, MYC expression is decreased. In some aspects, MYC expression is increased.

In some embodiments, the guide sequences are ribonucleic acid guide sequences as described herein. In some embodiments, the guide sequence is from about 10 base pairs to about 150 base pairs in length. In some embodiments, the one or more guide sequences comprise two or more guide sequences. In some embodiments, the one or more guide sequences comprise a sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS. 1-8.

The methods described herein can be used to modify or modulate one or more genomic sequences (e.g., MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51 promoter region CTCF binding site) in a variety of cells, which includes somatic cells, stem cells, mitotic or post-mitotic cells, neurons, fibroblasts, or zygotes. A cell, zygote, embryo, or post-natal mammal can be of vertebrate (e.g., mammalian) origin. In some aspects, the vertebrates are mammals or avians. Particular examples include primate (e.g., human), rodent (e.g., mouse, rat), canine, feline, bovine, equine, caprine, porcine, or avian (e.g., chickens, ducks, geese, turkeys) cells, zygotes, embryos, or post-natal mammals. In some embodiments, the cell, zygote, embryo, or post-natal mammal is isolated (e.g., an isolated cell; an isolated zygote; an isolated embryo). In some embodiments, a mouse cell, mouse zygote, mouse embryo, or mouse post-natal mammal is used. In some embodiments, a rat cell, rat zygote, rat embryo, or rat post-natal mammal is used. In some embodiments, a human cell, human zygote or human embryo is used. The methods described herein can be used to modify or modulate one or more genomic sequences (e.g., methylate or demethylate a promoter region CTCF binding site) in a mammal (e.g., a mouse, a human) in vivo.

Stem cells may include totipotent, pluripotent, multipotent, oligipotent and unipotent stem cells. Specific examples of stem cells include embryonic stem cells, fetal stem cells, adult stem cells, and induced pluripotent stem cells (iPSCs) (e.g., see U.S. Published Application Nos. 2010/0144031, 2011/0076678, 2011/0088107, 2012/0028821 all of which are incorporated herein by reference).

Somatic cells may be primary cells (non-immortalized cells), such as those freshly isolated from an animal, or may be derived from a cell line capable of prolonged proliferation in culture (e.g., for longer than 3 months) or indefinite proliferation (immortalized cells). Adult somatic cells may be obtained from individuals, e.g., human subjects, and cultured according to standard cell culture protocols available to those of ordinary skill in the art. Somatic cells of use in aspects of the invention include mammalian cells, such as, for example, human cells, non-human primate cells, or rodent (e.g., mouse, rat) cells. They may be obtained by well-known methods from various organs, e.g., skin, lung, pancreas, liver, stomach, intestine, heart, breast, reproductive organs, muscle, blood, bladder, kidney, urethra and other urinary organs, etc., generally from any organ or tissue containing live somatic cells. Mammalian somatic cells useful in various embodiments include, for example, fibroblasts, Sertoli cells, granulosa cells, neurons, pancreatic cells, epidermal cells, epithelial cells, endothelial cells, hepatocytes, hair follicle cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), macrophages, monocytes, mononuclear cells, cardiac muscle cells, skeletal muscle cells, etc.

In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a mouse cell. In some embodiments, the cell is a cancer cell as disclosed herein.

In some aspects, methylation of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51) is increased by about 2-fold, 2.5-fold, 2.7-fold, 3.0 fold, 3.5-fold, 4.0 fold or more. In some aspects, methylation of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51) is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 500%, 600% or more. In some aspects, methylation of a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51) is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, methylation of the promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) is increased or decreased by at least one site, at least two sites, at least three sites, at least five sites, at least ten sites, at least fifteen sites, at least twenty sites, at least twenty-five sites, or more. In some aspects, methylation of CpGs in the promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) is increased or decreased by at least one site, at least two sites, at least three sites, at least five sites, at least ten sites, at least fifteen sites, at least twenty sites, at least twenty-five sites, or more. Reporters of genomic methylation are described in U.S. application Ser. No. 15/078,851, which is incorporated herein by reference in its entirety. Any method known in the art may be used to measure genomic methylation and is not limited.

In some aspects, expression of a gene having a promoter region CTCF binding site is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, expression of a gene having a promoter region CTCF binding site is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200% or more. Methods of measuring gene expression are known in the art. Any method known in the art may be used to measure gene expression and is not limited.

In some aspects, methylation of the MYC promoter CTCF binding site is increased by about 2-fold, 2.5-fold, 2.7-fold, 3.0 fold, 3.5-fold, 4.0 fold or more. In some aspects, methylation of the MYC promoter CTCF binding site is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 500%, 600% or more. In some aspects, methylation of the MYC promoter CTCF binding site is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, methylation of the MYC promoter region CTCF binding site is increased or decreased by at least one site, at least two sites, at least three sites, at least five sites, at least ten sites, at least fifteen sites, at least twenty sites, at least twenty-five sites, or more. In some aspects, methylation of CpGs in the MYC promoter region CTCF binding site is increased or decreased by at least one site, at least two sites, at least three sites, at least five sites, at least ten sites, at least fifteen sites, at least twenty sites, at least twenty-five sites, or more.

In some aspects, MYC expression is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, MYC expression is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200% or more. Methods of measuring MYC expression are known in the art. Any method known in the art may be used to measure MYC expression and is not limited.

In some aspects, the invention is directed to a method of producing a nonhuman mammal carrying modifications in a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) comprising introducing into a zygote or an embryo a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity, and one or more guide sequences. The zygote or the embryo is maintained under conditions in which the guide sequence hybridizes to a portion of each of the one or more genomic sequences, and the catalytically inactive site specific nuclease fused to an effector domain either methylates or demethylates the genomic sequence, thereby producing an embryo having one or more modified genomic sequences. The embryo having one or more modified genomic sequences may be transferred into a foster nonhuman mammalian mother. The foster nonhuman mammalian mother is maintained under conditions in which one or more offspring carrying the one or more modified genomic sequences are produced, thereby producing a nonhuman mammal carrying modifications in one or more genomic sequences.

In some aspects, the invention is directed to a method of producing a nonhuman mammal carrying modifications in a MYC promoter CTCF binding site comprising introducing into a zygote or an embryo a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity, and one or more guide sequences. The zygote or the embryo is maintained under conditions in which the guide sequence hybridizes to a portion of each of the one or more genomic sequences (e.g., MYC promoter CTCF binding site), and the catalytically inactive site specific nuclease fused to an effector domain either methylates or demethylates the genomic sequence (e.g., MYC promoter CTCF binding site), thereby producing an embryo having one or more modified genomic sequences. The embryo having one or more modified genomic sequences (e.g., MYC promoter CTCF binding site) may be transferred into a foster nonhuman mammalian mother. The foster nonhuman mammalian mother is maintained under conditions in which one or more offspring carrying the one or more modified genomic sequences (e.g., MYC promoter CTCF binding site) are produced, thereby producing a nonhuman mammal carrying modifications in one or more genomic sequences (e.g., MYC promoter CTCF binding site).

As will be apparent to those of skill in the art, the nonhuman mammals can also be produced using methods described herein and/or with conventional methods, see for example, U.S. Published Application No. 2011/0302665. A method of producing a non-human mammalian embryo can comprise injecting non-human mammalian ES cells (e.g., iPSCs) into non-human tetraploid blastocysts and maintaining said resulting tetraploid blastocysts under conditions that result in formation of embryos, thereby producing a non-human mammalian embryo. In some embodiments, said non-human mammalian cells are mouse cells and said non-human mammalian embryo is a mouse. In some embodiments, said mouse cells are mutant mouse cells and are injected into said non-human tetraploid blastocysts by microinjection. In some embodiments laser-assisted micromanipulation or piezo injection is used. In some embodiments, a non-human mammalian embryo comprises a mouse embryo.

Another example of such conventional techniques is two step cloning which involves introducing embryonic stem (ES) and/or induced pluripotent stem (iPS) cells into a blastocyst (e.g., a tetraploid blastocyst) and maintaining the blastocyst under conditions that result in development of an embryo. The embryo is then transferred into an appropriate foster mother, such as a pseudopregnant female (e.g., of the same species as the embryo). The foster mother is then maintained under conditions that result in development of live offspring.

Another example is the use of the tetraploid complementation assay in which cells of two mammalian embryos are combined to form a new embryo (Tarn and Rossant, Develop, 750:6156-6163 (2003)). The assay involves producing a tetraploid cell in which every chromosome exists fourfold. This is done by taking an embryo at the two-cell stage and fusing the two cells by applying an electrical current. The resulting tetraploid cell continues to divide, and all daughter cells will also be tetraploid. Such a tetraploid embryo develops normally to the blastocyst stage and will implant in the wall of the uterus. In the tetraploid complementation assay, a tetraploid embryo (either at the morula or blastocyst stage) is combined with normal diploid embryonic stem cells (ES) from a different organism. The embryo develops normally; the fetus is exclusively derived from the ES cell, while the extraembryonic tissues are exclusively derived from the tetraploid cells.

Another conventional method used to produce nonhuman mammals includes pronuclear microinjection. DNA is introduced directly into the male pronucleus of a nonhuman mammal egg just after fertilization. Similar to the two-step cloning described above, the egg is implanted into a pseudopregnant female. Offspring are screened for the integrated transgene. Heterozygous offspring can be subsequently mated to generate homozygous animals.

A variety of nonhuman mammals can be used in the methods described herein. For example, the nonhuman mammal can be a rodent (e.g., mouse, rat, guinea pig, hamster), a nonhuman primate, a canine, a feline, a bovine, an equine, a porcine or a caprine.

In some aspects, various mouse strains and mouse models of human disease are used in conjunction with the methods of producing a nonhuman mammal carrying mutations or other modifications (e.g., altered methylation) in one or more target nucleic acid sequences described herein (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51 promoter region CTCF binding site). One of ordinary skill in the art appreciates the thousands of commercially and non-commercially available strains of laboratory mice for modeling human disease. Mice models exist for diseases such as cancer, cardiovascular disease, autoimmune diseases and disorders, inflammatory diseases, diabetes (type 1 and 2), neurological diseases, and other diseases. Examples of commercially available research strains include, and is not limited to, 11BHSD2 Mouse, GSK3B Mouse, 129-E Mouse HSD1 1B1 Mouse, AK Mouse Immortomouse®, Athymic Nude Mouse, LCAT Mouse, B6 Albino Mouse, Lox-1 Mouse, B6C3F1 Mouse, Ly5 Mouse, B6D2F1 (BDF1) Mouse, MMP9 Mouse, BALB/c Mouse, NIH-III Nude Mouse, BALB/c Nude Mouse, NOD Mouse, NOD SCID Mouse, Black Swiss Mouse, NSE-p25 Mouse, C3H Mouse, NU/NU Nude Mouse, C57BL/6-E Mouse, PCSK9 Mouse, C57BL/6N Mouse, PGP Mouse (P-glycoprotein Deficient), CB6F1 Mouse, repTOP™ ERE-Luc Mouse, CD-I® Mouse, repTOP™ mitolRE Mouse, CD-I® Nude Mouse, repTOP™ PPRE-Luc Mouse, CD1-E Mouse, Rip-HAT Mouse, CD2F1 (CDF1) Mouse, SCID Hairless Congenic (SHC™) Mouse, CF-1TM Mouse, SCID Hairless Outbred (SHO™) Mouse, DBA/2 Mouse, SJL-E Mouse, Fox Chase CB17™ Mouse, SKH1-E Mouse, Fox Chase SCID® Beige Mouse, Swiss Webster (CFW®) Mouse, Fox Chase SCID® Mouse, TARGATT™ Mouse, FVB Mouse, THE POUND MOUSE™, and GLUT 4 Mouse. Other mouse strains include BALB/c, C57BL/6, C57BL/10, C3H, ICR, CBA, A/J, NOD, DBA/1, DBA/2, MOLD, 129, HRS, MRL, NZB, NIH, AKR, SJL, NZW, CAST, KK, SENCAR, C57L, SAMR1, SAMP1, C57BR, and NZO.

In some aspects, the method of producing a nonhuman mammal carrying modifications in one or more genomic sequences (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51 promoter region CTCF binding site) further comprises mating one or more commercially and/or non-commercially available nonhuman mammal with the nonhuman mammal carrying modifications in one or more genomic sequences (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51 promoter region CTCF binding site) produced by the methods described herein. The invention is also directed to nonhuman mammals produced by the methods described herein.

As will be apparent to those of skill in the art, a variety of methods can be used to introduce nucleic acid and/or protein into a cell, zygote, embryo, and or mammal. Suitable methods include calcium phosphate or lipid-mediated transfection, electroporation, injection, and transduction or infection using a vector (e.g., a viral vector such as an adenoviral vector, lentiviral vector, or adeno-associated viral vector). In some aspects, the nucleic acid and/or protein is complexed with a vehicle, e.g., a cationic vehicle, that facilitates uptake of the nucleic acid and/or protein, e.g., via endocytosis.

The method described herein can further comprise isolating the cell or zygote produced by the methods. Thus, in some aspects, the invention is directed to a cell or zygote (an isolated cell or zygote) produced by the methods described herein. In some aspects, the disclosure provides a clonal population of cells harboring the modification(s), replicating cultures comprising cells harboring the modification(s) and cells isolated from the generated animals.

The methods described herein can further comprise crossing the generated animals with other animals harboring genetic modifications (optionally in same strain background) and/or having one or more phenotypes of interest (e.g., disease susceptibility—such as NOD mice). In addition, the methods may comprise modifying a cell, zygote, and/or animal from a strain that harbors one or more genetic modifications and/or has one or more phenotypes of interest (e.g., disease susceptibility). In some aspects, the genetic modifications are epigenetic modifications.

The methods described herein can further comprise assessing whether the one or more target nucleic acids have been modified and/or modulated using a variety of known methods.

In some embodiments methods described herein are used to produce multiple genetic modifications in a cell, zygote, embryo, or animal, wherein at least one of the genetic modifications methylates or demethylates a CTCF region binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) promoter region CTCF binding site, and at least one of the genetic modifications is in a different gene or genomic location. In some embodiments, a genetic modification includes epigenetic modifications. The resulting cell, zygote, embryo, or animal, or a cell, zygote, embryo, or animal generated therefrom, is analyzed. In some embodiments at least one of the genetic modifications may be conditional (e.g., the effect of the modification, such as gene methylation or demethylation, only becomes manifest under certain conditions, which are typically under control of the artisan). In some embodiments animals are permitted to develop at least to post-natal stage, e.g., to adult stage. The appropriate conditions for the modification to produce an effect (sometimes termed “inducing conditions”) are imposed, and the phenotype of the animal is subsequently analyzed. A phenotype may be compared to that of an unmodified animal or to the phenotype prior to the imposition of the inducing conditions.

Analysis may comprise any type of phenotypic analysis known in the art, e.g., examination of the structure, size, development, weight, or function, of any tissue, organ, or organ system (or the entire organism), analysis of behavior, activity of any biological pathway or process, level of any particular substance or gene product, etc. In some embodiments analysis comprises gene expression analysis, e.g., at the level of mRNA or protein. In some embodiments such analysis may comprise, e.g., use of microarrays (e.g., oligonucleotide microarrays, sometimes termed “chips”), high throughput sequencing (e.g., RNASeq), ChIP on Chip analysis, ChlPSeq analysis, etc. In some embodiments high content screening may be used, in which elements of high throughput screening may be applied to the analysis of individual cells through the use of automated microscopy and image analysis (see, e.g., Zanella et al, (2010). High content screening: seeing is believing. Trends Biotechnol. 28:237-245). In some embodiments analysis comprises quantitative analyses of components of cells such as spatio-temporal distributions of individual proteins, cytoskeletal structures, vesicles, and organelles, e.g., when contacted with test agents, e.g., chemical compounds. In some embodiments activation or inhibition of individual proteins and protein-protein interactions and/or changes in biological processes and cell functions may be assessed. A range of fluorescent probes for biological processes, functions, and cell components are available and may be used, e.g., with fluorescence microscopy. In some embodiments cells or animals generated according to methods herein may comprise a reporter, e.g., a fluorescent reporter or enzyme (e.g., a luciferase such as Gaussia, Renilla, or firefly luciferase) that, for example, reports on the expression or activity of particular genes. Such reporter may be fused to a protein, so that the protein or its activity is rendered detectable, optionally using a non-invasive detection means, e.g., an imaging or detection means such as PET imaging, MRI, fluorescence detection. Multiplexed genome editing according to the invention may allow installation of reporters for detection of multiple proteins, e.g., 2-20 different proteins, e.g., in a cell, tissue, organ, or animal, e.g., in a living animal.

Multiplexed genome editing or modification according to the present invention may be useful to determine or examine the biological role(s) and/or roles in disease of genes of unknown function. For example, discovery of synthetic effects caused by modifications in first and second genes (e.g., wherein one of the modifications comprises altered methylation of a CTCF region binding site of a gene) may pinpoint a genetic or biochemical pathway in which such gene(s) or encoded gene product(s) is involved.

In some embodiments it is contemplated to use, in methods described herein, cells or zygotes generated in or derived from animals produced in projects such as the International Knockout Mouse Consortium (IKMC), the website of which is http://www.knockoutmouse.org). In some embodiments it is contemplated to cross animals generated as described herein with animals generated by or available through the IKMC. For example, in some embodiments a mouse gene to be modified according to methods described herein is any gene from the Mouse Genome Informatics (MGI) database for which sequences and genome coordinates are available, e.g., any gene predicted by the NCBI, Ensembl, and Vega (Vertebrate Genome Annotation) pipelines for mouse Genome Build 37 (NCBI) or Genome Reference Consortium GRCm38.

In some embodiments, a gene or genomic location to be modified is included in a genome of a species for which a fully sequenced genome exists. Genome sequences may be obtained, e.g., from the UCSC Genome Browser (http://genome.ncsc.edu/index.html). For example, in some embodiments a human gene or sequence to be modified according to methods described herein may be found in Human Genome Build hg19 (Genome Reference Consortium). In some embodiments a gene is any gene for which a Gene ID has been assigned in the Gene Database of the NCBI (http://www.ncbi.nlm.nih.gov/gene). In some embodiments a gene is any gene for which a genomic, cDNA, mRNA, or encoded gene product (e.g., protein) sequence is available in a database such as any of those available at the National Center for Biotechnology Information (www.nchi.nih.gov) or Universal Protein Resource (www.uniprot.org). Databases include, e.g., GenBank, RefSeq, Gene, UniProtKB/SwissProt, UniProtKB/Tremb1, and the like.

In some embodiments animals generated according to methods described herein may be useful in the identification of candidate agents for treatment of disease and/or for testing agents for potential toxicity or side effects. In some embodiments any method described herein may comprise contacting an animal generated according to methods described herein, e.g., any genetically modified animal generated as described herein, with a test agent (e.g., a small molecule, nucleic acid, polypeptide, lipid, etc.). In some embodiments contacting comprises administering the test agent. Administration may be by any route (e.g., oral, intravenous, intraperitoneal, gavage, topical, transdermal, intramuscular, enteral, subcutaneous), may be systemic or local, may include any dose (e.g., from about 0.01 mg/kg to about 500 mg/kg), may involve a single dose or multiple doses. In some embodiments a method may further comprise analyzing the animal. Such analysis may, for example assess the effect of the test agent in an animal having a genetic modification(s) introduced according to the methods. In some embodiments a test agent that reduces or enhances an effect of one or more genetic modification(s) may be identified. In some embodiments if a test agent reduces or inhibits development of a disease associated with or produced by the genetic modification(s), (or reduces or inhibits one or more symptoms or signs of such a disease) the test agent may be identified as a candidate agent for treatment of a disease associated with or produced by the genetic modification(s) or associated with or produced by naturally occurring mutations in a gene or genomic location harboring the genetic modification.

The term “small molecule” refers to an organic molecule that is less than about 2 kilodaltons (kDa) in mass. In some embodiments, the small molecule is less than about 1.5 kDa, or less than about 1 kDa. In some embodiments, the small molecule is less than abou t 800 daltons (Da), 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule contains multiple carbon-carbon bonds and can comprise one or more heteroatoms and/or one or more functional groups important for structural interaction with proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least two functional groups. Small molecules often comprise one or more cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups. In some embodiments a small molecule is an artificial (non-naturally occurring) molecule. In some embodiments, a small molecule is non-polymeric. In some embodiments, a small molecule is not an amino acid. In some embodiments, a small molecule is not a nucleotide. In some embodiments, a small molecule is not a saccharide. In some embodiments, the term “small molecule” excludes molecules that are ingredients found in standard tissue culture medium.

In some embodiments a cell may be a diseased cell or may originate from a subject suffering from a disease, e.g., a disease affecting the cell or organ from which the cell was obtained. In some embodiments a mutation is introduced into a genomic region of the cell that is associated with a disease (e.g., any disease of interest, such as diseases mentioned herein). For example, in some embodiments it is of interest to methylate or demethylate a gene or genomic location (e.g., a Promoter region CTCF binding site of a gene, a MYC Promoter CTCF binding site) that is known or suspected to be involved in disease pathogenesis and/or known or suspected to be associated with increased or decreased risk of developing a disease or particular manifestation(s) of a disease. In some embodiments it is of interest to methylate or demethylate a gene or genomic location (e.g., a Promoter region CTCF binding site of a gene, a MYC Promoter CTCF binding site) and determine whether such modification alters the risk of developing a disease or one or more manifestations of a disease, alters progression of the disease, or alters the response of a subject to therapy or candidate therapy for a disease. In some embodiments it is of interest to modify an abnormal or disease-associated nucleotide or sequence (e.g., a Promoter region CTCF binding site of a gene, a MYC Promoter CTCF binding site) to one that is normal or not associated with disease. In some embodiments this may allow production of genetically matched cells or cell lines (e.g., iPS cells or cell lines) that differ only at one or more selected sites of genetic modification (e.g., a Promoter region CTCF binding site of a gene, a MYC Promoter CTCF binding site). Multiplexed genome editing as described herein may allow for production of cells or cell lines that are isogenic except with regard to, e.g., between 2 and 20 selected sites of genetic alterations (e.g., within a Promoter region CTCF binding site of a gene, within a MYC Promoter CTCF binding site). This may allow for the study of the combined effect of multiple modifications that are suspected of or known to play a role in disease risk, development or progression.

Also disclosed herein are methods of modulating the expression of a gene with a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) in a subject in need thereof comprising introducing into the subject a catalytically inactive site specific nuclease fused to an effector domain having methylation or demethylation activity; and one or more guide sequences homologous or complementary to at least a portion of the promoter region CTCF binding site, thereby modulating the expression of the gene in cells of the subject. In some embodiments, the effector domain has methylation activity and the expression of a gene with a promoter region CTCF binding site is decreased. In some embodiments, the effector domain has methylation activity and the expression of a gene with a promoter region CTCF binding site is increased. In some embodiments, the effector domain has demethylation activity and the expression of a gene with a promoter region CTCF binding site is increased. In some embodiments, the effector domain has methylation activity and the expression of a gene with a promoter region CTCF binding site is decreased. In some embodiments, the effector domain has demethylation activity and the expression of a gene with a promoter region CTCF binding site is decreased. In some embodiments, the effector domain has methylation activity and MYC expression is decreased. In some embodiments, the effector domain has demethylation activity and MYC expression is increased.

In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas protein as described herein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cas9 protein as described herein. In some embodiments, the catalytically inactive site specific nuclease is a catalytically inactive Cpf1 protein as described herein. In some embodiments, the effector domain having methylation activity is DMNT3A-3L as described herein.

In some embodiments, the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DMNT3A-3L or dCas9-DMNT3A-3L without a 5′ NLS as described herein.

In some embodiments, the guide sequences are ribonucleic acid guide sequences as described herein. In some embodiments, the guide sequence is from about 10 base pairs to about 150 base pairs in length. In some embodiments, the one or more guide sequences comprise two or more guide sequences.

In some embodiments, the one or more guide sequences comprise a sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS. 1-8.

In some aspects, expression of a gene having a promoter region CTCF binding site is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, expression of a gene having a promoter region CTCF binding site is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200% or more. In some aspects, MYC expression is decreased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, MYC expression is increased by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200% or more.

In some aspects, the effector domain comprises DNMT3A-3L. In some aspects, the catalytically inactive site specific nuclease is a catalytically inactive Cas (e.g., Cas9). In some aspects, the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DNMT3A-3L or dCas9-DMNT3A-3L without a 5′ NLS.

Some aspects of the disclosure are related to methods of treating a subject in need thereof, comprising administering to the subject a composition that enhances, suppresses, reduces or eliminates the binding of CTCF to a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51). Also disclosed herein are methods of treating a disease or condition involving aberrant MYC expression in a subject, comprising administering to the subject a composition that modulates the binding of CTCF to a MYC promoter CTCF binding site. In some aspects, aberrant MYC expression is over-expression. In some aspects, aberrant MYC expression is under-expression. In some aspects, the subject has cancer.

In some aspects, the composition is a composition described herein. In some aspects, the composition comprises a nucleic acid sequence, protein, organic molecule, inorganic molecule, or small molecule. In some aspects, the composition reduces binding of CTCF to a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TGIF1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table 51) by about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In some aspects, composition increases binding of CTCF to a promoter region CTCF binding site of a gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 500%, 600% or more. In some aspects the subject is human. The disease or condition involving aberrant gene (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) expression in a subject is any disease or condition described herein. In some aspects the disease or condition is cancer as described herein. In some aspects, the cancer is colorectal cancer, leukemia or breast cancer.

Also disclosed herein are methods of screening for a compound that modulates expression of a gene having a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) comprising, contacting a cell with a test agent; and measuring methylation in the promoter region CTCF binding site, wherein the test agent is identified as a compound that modulates expression of the gene if the level of methylation of the promoter region CTCF binding site in the cell contacted with the test agent differs from the level of methylation of said promoter region CTCF binding site in a control cell not contacted with the test agent. In some aspects, the test agent is identified as an anti-cancer compound if the level of methylation of the promoter region CTCF binding site in the cell contacted with the test agent is higher than the level of methylation of said promoter region CTCF binding site in a control cell not contacted with the test agent.

Also disclosed herein are methods of screening for a compound that modulates MYC expression comprising, contacting a cell with a test agent; and measuring methylation in a MYC promoter CTCF binding site, wherein the test agent is identified as a compound that modulates MYC expression if the level of methylation of the MYC promoter CTCF binding site in the cell contacted with the test agent differs from the level of methylation of said MYC promoter CTCF binding site in a control cell not contacted with the test agent. In some aspects, the test agent is identified as an anti-cancer compound if the level of methylation of the MYC promoter CTCF binding site in the cell contacted with the test agent is higher than the level of methylation of said MYC promoter CTCF binding site in a control cell not contacted with the test agent.

Methods of measuring methylation are known in the art and are not limited. In some embodiments the cells used in the method comprise cancer cells.

Also disclosed herein are methods of screening for a compound that modulates expression of a gene having a promoter region CTCF binding site (e.g., an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1) comprising contacting a cell with a test agent; and measuring binding between CTCF protein and CTCF binding site(s) within the promoter region, wherein the test agent is identified as a compound that modulates expression of the gene if the level of binding between CTCF protein and CTCF binding site(s) within the promoter region in the cell contacted with the test agent differs from the level of binding in a control cell not contacted with the test agent.

Also disclosed herein are methods of screening for a compound that modulates MYC expression comprising contacting a cell with a test agent; and measuring binding between CTCF protein and CTCF binding site(s) within the MYC promoter, wherein the test agent is identified as a compound that modulates MYC expression if the level of binding between CTCF protein and CTCF binding site(s) within the MYC promoter in the cell contacted with the test agent differs from the level of binding in a control cell not contacted with the test agent.

Also disclosed herein are methods of screening for a compound that modulates MYC expression comprising, contacting a cell with a test agent; and measuring binding of CTCF to the MYC promoter CTCF binding site, wherein the test agent is identified as a compound that modulates MYC expression if the level of binding of CTCF to the MYC promoter CTCF binding site in the cell contacted with the test agent differs from the level of binding of CTCF to the MYC promoter CTCF binding site in a control cell not contacted with the test agent. In some aspects, the test agent is identified as an anti-cancer compound if the level of binding of CTCF to the MYC promoter CTCF binding site in the cell contacted with the test agent is lower than the level of binding of CTCF to the MYC promoter CTCF binding site in a control cell not contacted with the test agent. Methods of measuring binding of CTCF to a DNA site of interest are known in the art and are not limited. For example, one could use ChIP-Seq. In some embodiments the cells used in the method comprise cancer cells.

One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details of the description and the examples herein are representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

The articles “a” and “an” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. It is contemplated that all embodiments described herein are applicable to all different aspects of the invention where appropriate. It is also contemplated that any of the embodiments or aspects can be freely combined with one or more other such embodiments or aspects whenever appropriate. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. For example, any one or more nucleic acids, polypeptides, cells, species or types of organism, disorders, subjects, or combinations thereof, can be excluded.

Where the claims or description relate to a composition of matter, e.g., a nucleic acid, polypeptide, cell, or non-human transgenic animal, it is to be understood that methods of making or using the composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where the claims or description relate to a method, e.g., it is to be understood that methods of making compositions useful for performing the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by “about” or “approximately”, the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by “about” or “approximately”, the invention includes an embodiment in which the value is prefaced by “about” or “approximately”. “Approximately” or “about” generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value). It should be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. It should also be understood that unless otherwise indicated or evident from the context, any product or composition described herein may be considered “isolated.”

Specific examples of these methods are set forth below in the Examples.

EXAMPLES

Proper regulation of gene expression is dependent on specific interactions between enhancers and promoters, but the mechanisms responsible for this specificity are not well-understood. We have identified a class of human genes that utilize CTCF-CTCF interactions to connect different cell-type specific enhancers with a single promoter-proximal element that functions as a docking site for those enhancers. At these genes, the enhancers are often bound by CTCF in a cell-type specific fashion whereas the promoter-proximal enhancer-docking sites are constitutively bound by CTCF. The proto-oncogene MYC, which is controlled by different cell-type specific enhancers during development, is a prominent example of a gene regulated in this fashion. We find that many human cancer cells acquire super-enhancers at the MYC locus and exploit this CTCF-mediated enhancer-docking mechanism to express MYC at oncogenic levels. Genetic and epigenetic perturbation of the MYC enhancer-docking site in tumor cells reduces CTCF binding, super-enhancer interaction, MYC gene expression and cell proliferation. Additional genes with roles in cancer employ a CTCF-bound enhancer-docking site to engender interactions with tumor specific CTCF-bound enhancers. Thus, a CTCF-dependent enhancer-docking mechanism, which facilitates interaction with cell-specific enhancers during development, is exploited by cancer cells to dysregulate expression of prominent oncogenes. Oncogene enhancer-docking sites can be repressed by dCas9-DNMT—mediated DNA methylation and may thus represent a common vulnerability in multiple human cancers.

CTCF does not generally occupy enhancer and promoter elements (Cuddapah et al., 2009; Dixon et al., 2012; Handoko et al., 2011; Ji et al., 2016; Kim et al., 2007; Parelho et al., 2008; Phillips-Cremins et al., 2013; Rao et al., 2014; Rubio et al., 2008; Tang et al., 2015; Wendt et al., 2008), but where CTCF does bind these elements, it may engender enhancer-promoter interactions (Guo et al., 2015; Lee et al., 2017; Splinter et al., 2006; de Wit et al., 2015). This consideration led us to further investigate the class of human genes that contain CTCF-bound sites at promoters to learn whether these are utilized to facilitate contacts with enhancers via CTCF-CTCF interactions. We report here that 2000 human genes have highly conserved promoter-proximal sites that are bound by CTCF regardless of the cell type examined and that these sites can form contacts with diverse cell-type specific enhancers. These genes appear to have evolved CTCF enhancer-docking sites in order to facilitate contacts with the diverse CTCF-bound enhancers formed by cell-type specific transcription factors during development, and thus experience activation in a broad range of cell types. Interestingly, this set of genes with CTCF-bound enhancer-docking sites includes many important cancer-associated genes, and the enhancer-docking site of one of these, MYC, was studied in detail. Genetic and epigenetic perturbation of the MYC enhancer-docking site reduces CTCF binding, super-enhancer interaction, MYC gene expression and tumor cell proliferation. These observations reveal a mechanism for enhancer-promoter interaction that is employed during development to allow genes to have cell-specific contacts with diverse enhancers and is exploited by cancer cells to facilitate oncogenic expression of genes driven by diverse super-enhancers.

Example 1—Promoter-Proximal CTCF-Bound Sites are Putative Enhancer-Docking Sites

To gain insights into the interactions between genes and their regulatory elements that may be mediated by CTCF, we focused our study on genes that have CTCF-bound sites at their promoters and are expressed in multiple cell types through the activity of different cell-specific enhancers (FIG. 1A). Because genes and their regulatory elements generally interact within the context of large CTCF-mediated DNA loops (insulated neighborhoods) that encompass them and facilitate accurate assessment of enhancer-promoter interactions (Ji et al., 2016; Hnisz et al., 2016a), we selected for further analysis the 2148 genes that have promoter-proximal CTCF-bound sites and occur within constitutive insulated neighborhoods (CTCF-CTCF loops shared by all at least two out of three cells examined). These genes shared two prominent features. They tend to be expressed in multiple cell types, apparently through the action of different cell-specific enhancers (1725/2148 have evidence for cell-type specific enhancers within their neighborhoods and, where DNA interaction data is available, there is evidence for cell-type specific interactions between those enhancers and the promoter-proximal CTCF-bound sites). As a class, they include many genes with cancer-associated functions, including proto-oncogenes and genes involved in growth control (FIG. 1A, Table S1). Prominent among these was the MYC oncogene, which because of its role in a broad spectrum of cancer cells, we chose for further study of promoter-proximal CTCF sites that may play a role in enhancer-promoter looping.

TABLE S1 Constitutive CTCF peaks within a constitutive IN across HCT116, Jurkat, K562 within 2.5 kb of TSS loops to differential chr start stop gene enhancers chr9 116161118 116166118 NM_000031 yes chr19 45406506 45411506 NM_000041 yes chr11 108091059 108096059 NM_000051 yes chr1 57429313 57434313 NM_000066 yes chr1 201079194 201084194 NM_000069 yes chr11 2904495 2909495 NM_000076 yes chr10 104594790 104599790 NM_000102 yes chr5 149337800 149342800 NM_000112 yes chr19 11492518 11497518 NM_000121 yes chr1 169553269 169558269 NM_000130 yes chr1 24192359 24197359 NM_000147 yes chr17 73758780 73763780 NM_000154 yes chr3 50226543 50231543 NM_000172 yes chr5 142780754 142785754 NM_000176 yes chr20 33541120 33546120 NM_000178 yes chr19 10379017 10384017 NM_000201 yes chr21 35882073 35887073 NM_000219 yes chr2 27307111 27312111 NM_000221 yes chr16 67975515 67980515 NM_000229 yes chr11 47371753 47376753 NM_000256 yes chr1 171619273 171624273 NM_000261 yes chr17 49228420 49233420 NM_000269 yes chr18 21164081 21169081 NM_000271 yes chr10 102502968 102507968 NM_000278 yes chr1 161133681 161138681 NM_000309 yes chr11 62377713 62382713 NM_000327 yes chr6 16759221 16764221 NM_000332 yes chr1 201344328 201349328 NM_000364 yes chr1 45475305 45480305 NM_000374 yes chr6 36643956 36648956 NM_000389 yes chr17 40702076 40707076 NM_000413 yes chr7 142657003 142662003 NM_000420 yes chr1 26322148 26327148 NM_000437 yes chr19 17980282 17985282 NM_000453 yes chr12 56388543 56393543 NM_000456 yes chr1 173884016 173889016 NM_000488 yes chr5 37837282 37842282 NM_000514 yes chr17 61993712 61998712 NM_000515 yes chr11 17407706 17412706 NM_000525 yes chr3 135966667 135971667 NM_000532 yes chr17 7588368 7593368 NM_000546 yes chr20 34023470 34028470 NM_000557 yes chr1 154375169 154380169 NM_000565 yes chr1 207492317 207497317 NM_000574 yes chr5 131393847 131398847 NM_000588 yes chr5 140010535 140015535 NM_000591 yes chr7 22764261 22769261 NM_000600 yes chr7 150685644 150690644 NM_000603 yes chr11 6459754 6464754 NM_000613 yes chr1 169678343 169683343 NM_000655 yes chr7 100491092 100496092 NM_000665 yes chr3 52014800 52019800 NM_000666 yes chr15 78911137 78916137 NM_000743 yes chr15 78931087 78936087 NM_000750 yes chr8 67088380 67093380 NM_000756 yes chr17 38169114 38174114 NM_000759 yes chr7 91761340 91766340 NM_000786 yes chr17 61551922 61556922 NM_000789 yes chr2 171670700 171675700 NM_000817 yes chr17 72853507 72858507 NM_000835 yes chr6 2997550 3002550 NM_000904 yes chr1 153648664 153653664 NM_000906 yes chr12 120763092 120768092 NM_000928 yes chr11 64016495 64021495 NM_000932 yes chr2 68477151 68482151 NM_000945 yes chr6 57179915 57184915 NM_000947 yes chr19 14583674 14588674 NM_000955 yes chr1 93295094 93300094 NM_000969 yes chr1 24015769 24020769 NM_000975 yes chr19 17968187 17973187 NM_000980 yes chr19 11543578 11548578 NM_001001329 yes chr5 156770229 156775229 NM_001001343 yes chr17 40167094 40172094 NM_001001349 yes chr22 31029302 31034302 NM_001001479 yes chr11 19136192 19141192 NM_001001483 yes chr12 109528793 109533793 NM_001001655 yes chr21 36258487 36263487 NM_001001890 yes chr19 1239249 1244249 NM_001001975 yes chr12 54068012 54073012 NM_001002031 yes chr2 64748939 64753939 NM_001002243 yes chr5 115174803 115179803 NM_001002924 yes chr1 29447921 29452921 NM_001003682 yes chr22 30819111 30824111 NM_001003704 yes chr3 51973821 51978821 NM_001003931 yes chr9 113097664 113102664 NM_001003936 yes chr15 40395787 40400787 NM_001003942 yes chr1 206806381 206811381 NM_001004023 yes chr1 57282869 57287869 NM_001004303 yes chr7 100059394 100064394 NM_001004323 yes chr1 113260689 113265689 NM_001004440 yes chr2 220406228 220411228 NM_001005209 yes chr1 204795282 204800282 NM_001005388 yes chr1 149821681 149826681 NM_001005464 yes chr1 149810265 149815265 NM_001005464_2 yes chr2 159310892 159315892 NM_001005476 yes chr1 119908899 119913899 NM_001005783 yes chr1 154295536 154300536 NM_001005855 yes chr12 56471392 56476392 NM_001005915 yes chr1 93424579 93429579 NM_001006605 yes chr17 72730856 72735856 NM_001006638 yes chr1 207625145 207630145 NM_001006658 yes chr3 183964813 183969813 NM_001006941 yes chr11 64124125 64129125 NM_001006944 yes chr11 2168333 2173333 NM_001007139 yes chr1 174966071 174971071 NM_001007214 yes chr1 161065681 161070681 NM_001007255 yes chr1 115298171 115303171 NM_001007553 yes chr12 49728471 49733471 NM_001008223 yes chr7 135192375 135197375 NM_001008225 yes chr18 43751488 43756488 NM_001008239 yes chr6 34214385 34219385 NM_001008703 yes chr20 5091233 5096233 NM_001009923 yes chr1 109100471 109105471 NM_001010883 yes chr1 110690636 110695636 NM_001010898 yes chr3 195619932 195624932 NM_001010938 yes chr20 49408931 49413931 NM_001010974 yes chr8 71579100 71584100 NM_001011720 yes chr1 156828171 156833171 NM_001012331 yes chr9 35826244 35831244 NM_001012446 yes chr19 56162916 56167916 NM_001012478 yes chr10 27147516 27152516 NM_001012750 yes chr19 12073369 12078369 NM_001012753 yes chr1 145586935 145591935 NM_001012758 yes chr16 67698217 67703217 NM_001012984 yes chr2 25013751 25018751 NM_001013663 yes chr12 31942675 31947675 NM_001013699 yes chr16 67676530 67681530 NM_001013838 yes chr5 43037947 43042947 NM_001014279 yes chr2 71556385 71561385 NM_001014972 yes chr17 4689754 4694754 NM_001014985 yes chr17 73519283 73524283 NM_001015002 yes chrX 106957211 106962211 NM_001015881 yes chr1 202160618 202165618 NM_001017403 yes chr6 117921205 117926205 NM_001017408 yes chr1 249197942 249202942 NM_001017434 yes chr17 61521045 61526045 NM_001017916 yes chr22 21981840 21986840 NM_001017964 yes chr1 91867926 91872926 NM_001017975 yes chr22 30780802 30785802 NM_001017981 yes chr1 155160206 155165206 NM_001018016 yes chr22 21316918 21321918 NM_001018060 yes chr5 142781545 142786545 NM_001018076 yes chr9 130562960 130567960 NM_001018078 yes chr17 49228397 49233397 NM_001018136 yes chr17 49240296 49245296 NM_001018137 yes chr17 49241583 49246583 NM_001018138 yes chr17 49241362 49246362 NM_001018139 yes chr1 154242539 154247539 NM_001018837 yes chr3 183733248 183738248 NM_001023587 yes chr1 153597617 153602617 NM_001024211 yes chr1 153597244 153602244 NM_001024212 yes chr1 153597024 153602024 NM_001024213 yes chr5 148928615 148933615 NM_001025105 yes chr21 44525188 44530188 NM_001025203 yes chr6 43735446 43740446 NM_001025366 yes chr1 161037260 161042260 NM_001025598 yes chr19 18109441 18114441 NM_001025604 yes chr12 56433186 56438186 NM_001029 yes chr8 101155599 101160599 NM_001029860 yes chr1 27927505 27932505 NM_001029882 yes chr5 176825137 176830137 NM_001029886 yes chr12 51661702 51666702 NM_001031628 yes chr5 176728245 176733245 NM_001031677 yes chr3 47552699 47557699 NM_001031703 yes chr19 30203463 30208463 NM_001031726 yes chr1 168145583 168150583 NM_001031800 yes chr8 103663692 103668692 NM_001032282 yes chr12 98906851 98911851 NM_001032283 yes chr20 48727235 48732235 NM_001032288 yes chr1 40365187 40370187 NM_001033081 yes chr17 27276008 27281008 NM_001033561 yes chr12 121075922 121080922 NM_001033677 yes chr12 51661494 51666494 NM_001033873 yes chr12 56507874 56512874 NM_001035267 yes chr12 120905160 120910160 NM_001037494 yes chr22 30683116 30688116 NM_001037666 yes chr1 151029625 151034625 NM_001038707 yes chr7 37022217 37027217 NM_001039459 yes chr11 63603900 63608900 NM_001039469 yes chr11 64065363 64070363 NM_001039496 yes chr1 155291438 155296438 NM_001039517 yes chr1 110750836 110755836 NM_001039574 yes chr14 23396294 23401294 NM_001039619 yes chr15 91470910 91475910 NM_001039675 yes chr14 24896231 24901231 NM_001039771 yes chr15 90317089 90322089 NM_001039958 yes chr7 1123943 1128943 NM_001039966 yes chr17 7530912 7535912 NM_001040 yes chr5 140010786 140015786 NM_001040021 yes chr12 32906387 32911387 NM_001040436 yes chr5 159623548 159628548 NM_001040442 yes chr8 80677598 80682598 NM_001040708 yes chr17 7182554 7187554 NM_001042 yes chr19 7743207 7748207 NM_001042461 yes chrX 14045535 14050535 NM_001042479 yes chr16 29815358 29820358 NM_001042539 yes chr3 196666964 196671964 NM_001042540 yes chr9 35826722 35831722 NM_001042589 yes chr1 113247525 113252525 NM_001042678 yes chr1 113247178 113252178 NM_001042679 yes chr4 110352371 110357371 NM_001042734 yes chr9 127613196 127618196 NM_001045476 yes chr1 28842245 28847245 NM_001048194 yes chr19 4906942 4911942 NM_001048201 yes chr17 38571702 38576702 NM_001067 yes chr6 3155283 3160283 NM_001069 yes chr20 33461828 33466828 NM_001076552 yes chr17 26217909 26222909 NM_001076680 yes chr17 42295750 42300750 NM_001076683 yes chr17 42294541 42299541 NM_001076684 yes chr16 68295919 68300919 NM_001076785 yes chr9 99326703 99331703 NM_001077181 yes chr2 178970566 178975566 NM_001077197 yes chr11 65147645 65152645 NM_001077241 yes chr10 104151835 104156835 NM_001077494 yes chr7 73079674 73084674 NM_001077621 yes chr6 44188741 44193741 NM_001078175 yes chr6 44188863 44193863 NM_001078177 yes chr11 67234248 67239248 NM_001078650 yes chr5 140996122 141001122 NM_001079812 yes chr2 202095666 202100666 NM_001080124 yes chr17 73778420 73783420 NM_001080419 yes chr19 6735133 6740133 NM_001080452 yes chr2 176946190 176951190 NM_001080458 yes chr17 72355458 72360458 NM_001080466 yes chr1 156861023 156866023 NM_001080471 yes chr20 42937392 42942392 NM_001080472 yes chr7 134231349 134236349 NM_001080538 yes chr9 139255763 139260763 NM_001080849 yes chr12 120701074 120706074 NM_001080855 yes chr19 12938730 12943730 NM_001080997 yes chr19 46281361 46286361 NM_001081563 yes chr17 63130956 63135956 NM_001081955 yes chr20 62336294 62341294 NM_001083113 yes chr9 130495128 130500128 NM_001085347 yes chr17 27914110 27919110 NM_001085454 yes chr16 2388247 2393247 NM_001089 yes chr10 81368195 81373195 NM_001093770 yes chr12 50448920 50453920 NM_001095 yes chr7 1125223 1130223 NM_001098201 yes chr17 1957104 1962104 NM_001098202 yes chr10 43902196 43907196 NM_001098204 yes chr10 43901832 43906832 NM_001098205 yes chr10 43900799 43905799 NM_001098206 yes chr12 48150389 48155389 NM_001098531 yes chr12 48149681 48154681 NM_001098532 yes chr12 50133092 50138092 NM_001098576 yes chr11 10671348 10676348 NM_001098579 yes chr12 53843386 53848386 NM_001098620 yes chr10 81317717 81322717 NM_001098668 yes chr12 52461258 52466258 NM_001098673 yes chr11 65337320 65342320 NM_001098784 yes chr14 24709380 24714380 NM_001099274 yes chr8 145200419 145205419 NM_001099280 yes chr2 232570735 232575735 NM_001099285 yes chr12 56658567 56663567 NM_001099337 yes chr11 65341009 65346009 NM_001099409 yes chr15 75133052 75138052 NM_001099436 yes chr8 86130151 86135151 NM_001099670 yes chr20 43989240 43994240 NM_001099791 yes chr19 12883934 12888934 NM_001100176 yes chr4 39977076 39982076 NM_001100399 yes chr7 5567732 5572732 NM_001101 yes chr2 101765409 101770409 NM_001102426 yes chr14 21570363 21575363 NM_001102454 yes chr1 155288140 155293140 NM_001105203 yes chr1 155291228 155296228 NM_001105205 yes chr16 31467817 31472817 NM_001105247 yes chr1 10090541 10095541 NM_001105562 yes chr10 124893067 124898067 NM_001105574 yes chr1 156548657 156553657 NM_001105669 yes chr16 30032155 30037155 NM_001109659 yes chr8 146124346 146129346 NM_001109689 yes chr22 42333723 42338723 NM_001110215 yes chr1 55269236 55274236 NM_001110533 yes chr2 158482899 158487899 NM_001111032 yes chr4 39697164 39702164 NM_001111112 yes chr1 161006274 161011274 NM_001113205 yes chr1 163039195 163044195 NM_001113380 yes chr1 163036544 163041544 NM_001113381 yes chr1 201977190 201982190 NM_001114309 yes chr6 126109409 126114409 NM_001122842 yes chr16 31117115 31122115 NM_001122957 yes chr1 109653979 109658979 NM_001122961 yes chr9 127949718 127954718 NM_001123355 yes chr1 71510991 71515991 NM_001126044 yes chr19 35998891 36003891 NM_001126059 yes chr14 23282607 23287607 NM_001126105 yes chr14 23286520 23291520 NM_001126106 yes chr17 7576311 7581311 NM_001126115 yes chr18 710162 715162 NM_001126123 yes chr1 92946856 92951856 NM_001127215 yes chr1 92949128 92954128 NM_001127216 yes chr19 47733523 47738523 NM_001127240 yes chr14 50997300 51002300 NM_001127713 yes chr16 89677216 89682216 NM_001128141 yes chr1 95697211 95702211 NM_001128142 yes chr18 43301592 43306592 NM_001128588 yes chr15 42064132 42069132 NM_001128608 yes chr18 12655412 12660412 NM_001128626 yes chr15 40507129 40512129 NM_001128628 yes chr10 105236497 105241497 NM_001129742 yes chr8 145688531 145693531 NM_001129888 yes chr19 34285251 34290251 NM_001129994 yes chr5 141014017 141019017 NM_001130029 yes chr1 75196592 75201592 NM_001130042 yes chr11 47276968 47281968 NM_001130101 yes chr15 74419215 74424215 NM_001130136 yes chr15 74419496 74424496 NM_001130137 yes chr15 74420090 74425090 NM_001130138 yes chr6 135499953 135504953 NM_001130172 yes chr12 56580858 56585858 NM_001130420 yes chr1 225963015 225968015 NM_001130440 yes chr4 141346315 141351315 NM_001130675 yes chr3 187451785 187456785 NM_001130845 yes chr11 67270343 67275343 NM_001130848 yes chr17 46891969 46896969 NM_001130918 yes chr1 110034201 110039201 NM_001134400 yes chr1 110034158 110039158 NM_001134402 yes chr1 86041546 86046546 NM_001134445 yes chr6 41752681 41757681 NM_001134493 yes chr20 62336865 62341865 NM_001134758 yes chr1 159912886 159917886 NM_001135050 yes chr22 19935960 19940960 NM_001135161 yes chr11 72430903 72435903 NM_001135190 yes chr1 151168521 151173521 NM_001135636 yes chr17 1417682 1422682 NM_001135642 yes chr1 40040021 40045021 NM_001135653 yes chr17 43210514 43215514 NM_001135705 yes chr19 35604232 35609232 NM_001136007 yes chr1 225995276 226000276 NM_001136018 yes chr1 249150625 249155625 NM_001136036 yes chr14 24766539 24771539 NM_001136050 yes chr19 18041324 18046324 NM_001136203 yes chr1 44396492 44401492 NM_001136215 yes chr6 44235980 44240980 NM_001137560 yes chr16 67515216 67520216 NM_001138 yes chr11 61714856 61719856 NM_001139443 yes chr6 109701515 109706515 NM_001142401 yes chr19 45907107 45912107 NM_001142502 yes chr9 130522219 130527219 NM_001142531 yes chr9 130522197 130527197 NM_001142532 yes chr11 63271024 63276024 NM_001142535 yes chr11 63272959 63277959 NM_001142537 yes chr1 46710867 46715867 NM_001142548 yes chr1 156695763 156700763 NM_001142560 yes chr17 33444388 33449388 NM_001142571 yes chr17 74378190 74383190 NM_001142601 yes chr17 74378789 74383789 NM_001142602 yes chr7 86686514 86691514 NM_001142749 yes chr16 30994019 30999019 NM_001142777 yes chr17 6915556 6920556 NM_001142798 yes chr5 176736792 176741792 NM_001142935 yes chr6 134496510 134501510 NM_001143677 yes chr6 134494570 134499570 NM_001143678 yes chr15 91425776 91430776 NM_001143783 yes chr15 91425165 91430165 NM_001143785 yes chr12 56318409 56323409 NM_001143853 yes chr10 105003144 105008144 NM_001143909 yes chr1 28049990 28054990 NM_001143912 yes chr1 245131131 245136131 NM_001143943 yes chr17 37319914 37324914 NM_001143968 yes chr17 7586889 7591889 NM_001143990 yes chr17 7588258 7593258 NM_001143991 yes chr17 7589167 7594167 NM_001143992 yes chr15 89087434 89092434 NM_001144074 yes chr11 118475806 118480806 NM_001144758 yes chr17 40166683 40171683 NM_001144766 yes chr6 56816926 56821926 NM_001144769 yes chr1 117111215 117116215 NM_001144822 yes chr9 127903338 127908338 NM_001144877 yes chr17 40169587 40174587 NM_001144927 yes chr12 3859866 3864866 NM_001144958 yes chr11 119064084 119069084 NM_001145018 yes chr17 45916199 45921199 NM_001145023 yes chr20 33732661 33737661 NM_001145025 yes chr19 14167471 14172471 NM_001145028 yes chr17 28254374 28259374 NM_001145053 yes chr11 61273772 61278772 NM_001145077 yes chr5 157096061 157101061 NM_001145132 yes chr19 16175817 16180817 NM_001145160 yes chr2 175197321 175202321 NM_001145250 yes chr1 156022017 156027017 NM_001145264 yes chr17 38471973 38476973 NM_001145301 yes chr2 178126359 178131359 NM_001145412 yes chr3 38535263 38540263 NM_001145464 yes chr1 117661911 117666911 NM_001145635 yes chr1 45137894 45142894 NM_001145636 yes chr5 54466505 54471505 NM_001145734 yes chr5 150223585 150228585 NM_001145805 yes chr11 1858219 1863219 NM_001145829 yes chr11 1858932 1863932 NM_001145841 yes chr7 91761559 91766559 NM_001146152 yes chr1 43230255 43235255 NM_001146289 yes chr3 52006146 52011146 NM_001146314 yes chr12 6979949 6984949 NM_001146316 yes chr17 8645654 8650654 NM_001158261 yes chr1 120351703 120356703 NM_001159352 yes chr1 204910854 204915854 NM_001160331 yes chr17 27051449 27056449 NM_001160407 yes chr20 56193132 56198132 NM_001160417 yes chr20 32897108 32902108 NM_001161766 yes chr1 75196336 75201336 NM_001162916 yes chr17 73627014 73632014 NM_001162995 yes chr19 46193241 46198241 NM_001163377 yes chr9 115093444 115098444 NM_001163788 yes chr17 72730459 72735459 NM_001163989 yes chr1 186342390 186347390 NM_001164245 yes chr3 57991627 57996627 NM_001164317 yes chr12 110904026 110909026 NM_001164372 yes chr12 110903589 110908589 NM_001164373 yes chr7 150752552 150757552 NM_001164410 yes chr7 100610404 100615404 NM_001164462 yes chr19 35643345 35648345 NM_001164605 yes chr8 141643146 141648146 NM_001164623 yes chr12 53815139 53820139 NM_001164690 yes chr14 23538279 23543279 NM_001164816 yes chr1 35322146 35327146 NM_001164824 yes chr1 35322838 35327838 NM_001164825 yes chr17 37790833 37795833 NM_001165937 yes chr17 8019734 8024734 NM_001165960 yes chr17 8024910 8029910 NM_001165967 yes chr17 57295328 57300328 NM_001165993 yes chr11 102215413 102220413 NM_001166 yes chr11 67139148 67144148 NM_001166212 yes chr11 17408378 17413378 NM_001166290 yes chr3 50281826 50286826 NM_001166425 yes chr11 116660636 116665636 NM_001166598 yes chr4 87853654 87858654 NM_001166693 yes chr6 41701497 41706497 NM_001167827 yes chr12 123847256 123852256 NM_001167856 yes chr22 50962368 50967368 NM_001169109 yes chr22 50962074 50967074 NM_001169110 yes chr22 50961533 50966533 NM_001169111 yes chr9 33400180 33405180 NM_001170 yes chr1 86171616 86176616 NM_001170670 yes chr11 61246085 61251085 NM_001170753 yes chr10 98589517 98594517 NM_001170765 yes chr12 53642870 53647870 NM_001170790 yes chrX 70313499 70318499 NM_001170931 yes chr3 50605958 50610958 NM_001171741 yes chr6 35307835 35312835 NM_001171818 yes chr1 29446513 29451513 NM_001171868 yes chr1 33333914 33338914 NM_001171940 yes chr1 33335593 33340593 NM_001171941 yes chr12 122324017 122329017 NM_001171993 yes chr14 68084079 68089079 NM_001172 yes chr12 58174028 58179028 NM_001172695 yes chr1 29060952 29065952 NM_001172828 yes chr1 29060633 29065633 NM_001173128 yes chr12 53712912 53717912 NM_001173466 yes chr2 220323034 220328034 NM_001173476 yes chr10 73076510 73081510 NM_001174098 yes chr19 1809736 1814736 NM_001178002 yes chr12 57502696 57507696 NM_001178078 yes chr12 57501589 57506589 NM_001178079 yes chr10 104261219 104266219 NM_001178133 yes chr12 56519486 56524486 NM_001184796 yes chr3 14714106 14719106 NM_001184957 yes chr22 20116864 20121864 NM_001185024 yes chr1 161085362 161090362 NM_001185092 yes chr1 161085407 161090407 NM_001185093 yes chr1 161085408 161090408 NM_001185094 yes chr9 140119518 140124518 NM_001190228 yes chr5 37833429 37838429 NM_001190468 yes chr15 40572287 40577287 NM_001190479 yes chr20 30308401 30313401 NM_001191 yes chr16 67593810 67598810 NM_001191022 yes chr6 13572261 13577261 NM_001193267 yes chr19 46146275 46151275 NM_001193268 yes chr19 46144485 46149485 NM_001193269 yes chr1 205088650 205093650 NM_001193272 yes chr1 249150815 249155815 NM_001193328 yes chr2 65213079 65218079 NM_001193493 yes chr1 48460062 48465062 NM_001194986 yes chr7 128503902 128508902 NM_001195150 yes chr20 62336880 62341880 NM_001195653 yes chr2 220405319 220410319 NM_001195731 yes chr6 53407427 53412427 NM_001197115 yes chr20 43988077 43993077 NM_001197129 yes chr14 24801777 24806777 NM_001198568 yes chr3 39231587 39236587 NM_001198621 yes chr17 7459109 7464109 NM_001198622 yes chr17 49241133 49246133 NM_001198682 yes chr15 55698223 55703223 NM_001198784 yes chr1 167187566 167192566 NM_001198786 yes chr11 64946186 64951186 NM_001198868 yes chr11 64946650 64951650 NM_001198869 yes chr7 128500357 128505357 NM_001198909 yes chr1 24739087 24744087 NM_001199012 yes chr1 24737762 24742762 NM_001199013 yes chr3 128877573 128882573 NM_001199469 yes chr20 35231637 35236637 NM_001199534 yes chr12 56543704 56548704 NM_001199629 yes chr1 95580979 95585979 NM_001199691 yes chr7 150752787 150757787 NM_001199692 yes chr7 150757134 150762134 NM_001199693 yes chr7 150757229 150762229 NM_001199694 yes chr1 156673108 156678108 NM_001199723 yes chr11 64779085 64784085 NM_001199745 yes chr2 176991968 176996968 NM_001199746 yes chr2 176991922 176996922 NM_001199747 yes chr12 89916844 89921844 NM_001199777 yes chr12 89917539 89922539 NM_001199781 yes chr2 25013484 25018484 NM_001199803 yes chr1 212206502 212211502 NM_001199809 yes chr7 26329015 26334015 NM_001199835 yes chr7 26401445 26406445 NM_001199838 yes chr8 22547402 22552402 NM_001199880 yes chr8 22547593 22552593 NM_001199881 yes chr17 48169601 48174601 NM_001199898 yes chr17 48170559 48175559 NM_001199899 yes chr17 48170139 48175139 NM_001199900 yes chr9 32570682 32575682 NM_001199987 yes chr17 17397209 17402209 NM_001199989 yes chr1 182756084 182761084 NM_001200050 yes chr8 9006652 9011652 NM_001201329 yes chr2 171782448 171787448 NM_001201428 yes chr16 47175436 47180436 NM_001201477 yes chr1 206783404 206788404 NM_001201478 yes chr9 35094046 35099046 NM_001201484 yes chr1 110879445 110884445 NM_001201545 yes chr12 52414116 52419116 NM_001202233 yes chr9 132401948 132406948 NM_001202403 yes chr11 17370809 17375809 NM_001202439 yes chr7 101456684 101461684 NM_001202543 yes chr7 101456788 101461788 NM_001202546 yes chr1 27112179 27117179 NM_001202554 yes chr19 12248722 12253722 NM_001203250 yes chr1 111171596 111176596 NM_001204269 yes chr19 46847751 46852751 NM_001204284 yes chr17 75082225 75087225 NM_001204408 yes chr17 75082735 75087735 NM_001204410 yes chr20 3798671 3803671 NM_001204446 yes chr15 55698208 55703208 NM_001204450 yes chr19 49974966 49979966 NM_001204502 yes chr19 49975318 49980318 NM_001204503 yes chr10 103537626 103542626 NM_001206389 yes chr1 19809635 19814635 NM_001206540 yes chr1 87167753 87172753 NM_001206651 yes chr1 160066118 160071118 NM_001206665 yes chr11 65476973 65481973 NM_001206833 yes chr12 13246240 13251240 NM_001206843 yes chr7 100228773 100233773 NM_001206855 yes chr19 42634125 42639125 NM_001207025 yes chr19 50177909 50182909 NM_001207042 yes chr19 49147069 49152069 NM_001217 yes chr6 36643987 36648987 NM_001220778 yes chr4 110622129 110627129 NM_001226 yes chr1 160157785 160162785 NM_001231 yes chr20 33460266 33465266 NM_001242393 yes chr1 110524887 110529887 NM_001242673 yes chr12 52602139 52607139 NM_001242696 yes chr12 56612985 56617985 NM_001242826 yes chr11 47196176 47201176 NM_001242832 yes chr1 205195559 205200559 NM_001242925 yes chr6 20401410 20406410 NM_001243076 yes chr6 37135422 37140422 NM_001243186 yes chr17 19263551 19268551 NM_001243473 yes chr12 51439582 51444582 NM_001243689 yes chr11 119249936 119254936 NM_001243759 yes chr1 10488304 10493304 NM_001243768 yes chr12 54580278 54585278 NM_001243787 yes chr12 54580241 54585241 NM_001243789 yes chr20 62494081 62499081 NM_001243891 yes chr3 50652062 50657062 NM_001243925 yes chr3 50646793 50651793 NM_001243926 yes chr12 6807509 6812509 NM_001244014 yes chr12 6807096 6812096 NM_001244015 yes chr14 69258131 69263131 NM_001244698 yes chr14 69260460 69265460 NM_001244701 yes chr9 115092806 115097806 NM_001244898 yes chr3 49938570 49943570 NM_001244937 yes chr6 74017438 74022438 NM_001251874 yes chr1 200990328 200995328 NM_001252100 yes chr5 57753466 57758466 NM_001252226 yes chr17 73125390 73130390 NM_001252377 yes chr1 154972606 154977606 NM_001252406 yes chr1 155175272 155180272 NM_001252607 yes chr7 137684347 137689347 NM_001253775 yes chr1 114445045 114450045 NM_001253852 yes chr1 114445246 114450246 NM_001253853 yes chr4 154263301 154268301 NM_001253861 yes chr11 75477278 75482278 NM_001253891 yes chr7 130123516 130128516 NM_001253901 yes chr7 130123688 130128688 NM_001253902 yes chr11 60736613 60741613 NM_001254750 yes chr17 40727234 40732234 NM_001256014 yes chr17 40727349 40732349 NM_001256015 yes chr19 30203952 30208952 NM_001256046 yes chr19 30204196 30209196 NM_001256047 yes chr1 46804350 46809350 NM_001256127 yes chr11 102215605 102220605 NM_001256163 yes chr16 29799534 29804534 NM_001256269 yes chr16 29799789 29804789 NM_001256270 yes chr1 146711791 146716791 NM_001256336 yes chr1 54409499 54414499 NM_001256409 yes chr22 19163518 19168518 NM_001256534 yes chrX 153623906 153628906 NM_001256577 yes chr1 155143763 155148763 NM_001256599 yes chr1 155144730 155149730 NM_001256601 yes chr11 66244984 66249984 NM_001256670 yes chr15 74282189 74287189 NM_001256672 yes chr15 74284463 74289463 NM_001256676 yes chr19 19246810 19251810 NM_001256766 yes chr12 50464868 50469868 NM_001256830 yes chr11 67118567 67123567 NM_001256870 yes chr1 38155573 38160573 NM_001256875 yes chr12 50503264 50508264 NM_001257133 yes chr11 118659157 118664157 NM_001257191 yes chr17 4611289 4616289 NM_001257328 yes chr1 156093408 156098408 NM_001257374 yes chr17 73994487 73999487 NM_001258 yes chr16 22306196 22311196 NM_001258033 yes chr20 50156758 50161758 NM_001258296 yes chr11 44969259 44974259 NM_001258320 yes chr11 44970357 44975357 NM_001258321 yes chr11 44969955 44974955 NM_001258323 yes chr19 1063422 1068422 NM_001258328 yes chr17 48621950 48626950 NM_001258372 yes chr11 72143228 72148228 NM_001258392 yes chr5 139737287 139742287 NM_001258426 yes chr19 35737059 35742059 NM_001260489 yes chr9 130545805 130550805 NM_001261 yes chr12 122324137 122329137 NM_001261400 yes chr10 104152853 104157853 NM_001261403 yes chr1 94310206 94315206 NM_001261408 yes chr1 155021248 155026248 NM_001261464 yes chr17 56403652 56408652 NM_001261835 yes chr4 113555620 113560620 NM_001267039 yes chr14 52116076 52121076 NM_001267046 yes chr1 109654085 109659085 NM_001267048 yes chr20 62336705 62341705 NM_001267548 yes chr10 35413269 35418269 NM_001267562 yes chr10 35481555 35486555 NM_001267568 yes chr10 35482330 35487330 NM_001267570 yes chr7 100447841 100452841 NM_001267812 yes chr20 56098208 56103208 NM_001269040 yes chr20 56097423 56102423 NM_001269041 yes chr20 56097683 56102683 NM_001269043 yes chr20 56097663 56102663 NM_001269050 yes chr21 35881137 35886137 NM_001270402 yes chr21 35881113 35886113 NM_001270403 yes chr12 123752281 123757281 NM_001270433 yes chr12 123750301 123755301 NM_001270434 yes chr1 183602576 183607576 NM_001270439 yes chr19 12943742 12948742 NM_001270440 yes chr19 12942890 12947890 NM_001270441 yes chr19 12938989 12943989 NM_001270443 yes chr12 93961675 93966675 NM_001270467 yes chr12 93962257 93967257 NM_001270468 yes chr12 93962957 93967957 NM_001270469 yes chr12 93963802 93968802 NM_001270471 yes chr6 52147179 52152179 NM_001270472 yes chr6 138185825 138190825 NM_001270507 yes chr1 10487659 10492659 NM_001270517 yes chr1 201082000 201087000 NM_001270601 yes chr1 110571699 110576699 NM_001270768 yes chr6 166794001 166799001 NM_001270879 yes chr1 36612615 36617615 NM_001270894 yes chr18 59990020 59995020 NM_001270949 yes chr16 768642 773642 NM_001271285 yes chr12 54716374 54721374 NM_001271734 yes chr5 148927394 148932394 NM_001271742 yes chr5 176728263 176733263 NM_001271828 yes chr19 12033383 12038383 NM_001271848 yes chr11 67273158 67278158 NM_001271849 yes chr1 205323718 205328718 NM_001271863 yes chr17 56030184 56035184 NM_001271875 yes chr15 78421377 78426377 NM_001271888 yes chr19 42827261 42832261 NM_001271938 yes chr6 41700765 41705765 NM_001271943 yes chr6 41699639 41704639 NM_001271945 yes chr1 153955342 153960342 NM_001272038 yes chr3 48512242 48517242 NM_001272082 yes chr9 116353934 116358934 NM_001276262 yes chr16 29814917 29819917 NM_001276275 yes chr1 207223825 207228825 NM_001276320 yes chr12 109122119 109127119 NM_001276471 yes chr11 64876826 64881826 NM_001277233 yes chr5 115175048 115180048 NM_001277783 yes chr11 3874433 3879433 NM_001277961 yes chr5 37832424 37837424 NM_001278098 yes chr17 46045394 46050394 NM_001278197 yes chr17 46045822 46050822 NM_001278198 yes chr1 155291686 155296686 NM_001278230 yes chr11 65147357 65152357 NM_001278250 yes chr11 65148672 65153672 NM_001278251 yes chr11 119209093 119214093 NM_001278431 yes chr14 21535919 21540919 NM_001278529 yes chr9 127531089 127536089 NM_001278546 yes chr17 72854466 72859466 NM_001278553 yes chr12 110885716 110890716 NM_001278556 yes chr6 57084612 57089612 NM_001278666 yes chr6 57083759 57088759 NM_001278668 yes chr19 35627397 35632397 NM_001278717 yes chr19 35628038 35633038 NM_001278718 yes chr19 1266767 1271767 NM_001280 yes chr7 142550276 142555276 NM_001280794 yes chr11 61581175 61586175 NM_001281501 yes chr11 61581376 61586376 NM_001281502 yes chr1 26558144 26563144 NM_001281517 yes chr1 32279152 32284152 NM_001281987 yes chr17 28254746 28259746 NM_001282129 yes chr11 119036922 119041922 NM_001282143 yes chr19 45455342 45460342 NM_001282176 yes chr14 21536531 21541531 NM_001282211 yes chr17 46798030 46803030 NM_001282275 yes chr17 46798554 46803554 NM_001282276 yes chr7 150723009 150728009 NM_001282291 yes chr11 119036543 119041543 NM_001282358 yes chr2 209116682 209121682 NM_001282386 yes chr2 209116527 209121527 NM_001282387 yes chr16 3146818 3151818 NM_001282415 yes chr11 64644685 64649685 NM_001282444 yes chr11 64071199 64076199 NM_001282450 yes chr11 64070500 64075500 NM_001282451 yes chr6 57177103 57182103 NM_001282488 yes chr17 72916858 72921858 NM_001282489 yes chr19 11404315 11409315 NM_001282509 yes chr20 49545250 49550250 NM_001282531 yes chr17 55820190 55825190 NM_001282544 yes chr1 28412648 28417648 NM_001282560 yes chr3 50261620 50266620 NM_001282619 yes chr11 11860470 11865470 NM_001282659 yes chr9 128021573 128026573 NM_001282679 yes chr1 171215110 171220110 NM_001282693 yes chr8 80676210 80681210 NM_001282851 yes chr8 126441951 126446951 NM_001282985 yes chr1 159767801 159772801 NM_001284217 yes chr15 34633016 34638016 NM_001284292 yes chr15 40597674 40602674 NM_001284297 yes chr8 10694909 10699909 NM_001284356 yes chr17 1529169 1534169 NM_001284498 yes chr20 48804620 48809620 NM_001285878 yes chr11 62444089 62449089 NM_001286077 yes chr6 155052012 155057012 NM_001286188 yes chr1 212206395 212211395 NM_001286229 yes chr1 157960563 157965563 NM_001286349 yes chr1 114519513 114524513 NM_001286352 yes chr14 24766560 24771560 NM_001286367 yes chr1 161193229 161198229 NM_001286373 yes chr15 91473299 91478299 NM_001286451 yes chr6 3454293 3459293 NM_001286456 yes chr21 47741313 47746313 NM_001286476 yes chr21 47741271 47746271 NM_001286477 yes chr6 44222808 44227808 NM_001286509 yes chr6 44222589 44227589 NM_001286510 yes chr6 44223127 44228127 NM_001286511 yes chr6 35702309 35707309 NM_001286574 yes chr16 29872109 29877109 NM_001286585 yes chr6 36851140 36856140 NM_001286635 yes chr6 43421870 43426870 NM_001286655 yes chr6 43420862 43425862 NM_001286656 yes chr9 132368663 132373663 NM_001286796 yes chr2 220358577 220363577 NM_001286811 yes chr19 18116477 18121477 NM_001286826 yes chr9 124919687 124924687 NM_001286828 yes chr12 49295393 49300393 NM_001286957 yes chr19 18389966 18394966 NM_001286968 yes chr6 43737222 43742222 NM_001287044 yes chr15 74608382 74613382 NM_001287181 yes chr8 71579182 71584182 NM_001287260 yes chr20 3764919 3769919 NM_001287516 yes chr20 3773886 3778886 NM_001287519 yes chr10 104151367 104156367 NM_001288724 yes chr19 46280377 46285377 NM_001288765 yes chr16 31467094 31472094 NM_001288767 yes chr8 145201048 145206048 NM_001288814 yes chr19 6737193 6742193 NM_001288962 yes chr3 138310725 138315725 NM_001288964 yes chr17 48501019 48506019 NM_001288968 yes chr19 19751976 19756976 NM_001288998 yes chr1 25941459 25946459 NM_001289010 yes chr14 23449351 23454351 NM_001289097 yes chr19 11544061 11549061 NM_001289102 yes chr17 7514882 7519882 NM_001289114 yes chr5 133858840 133863840 NM_001289984 yes chr4 76647206 76652206 NM_001290049 yes chr19 4907605 4912605 NM_001290051 yes chr19 4907879 4912879 NM_001290052 yes chr3 50308050 50313050 NM_001290062 yes chr14 24895992 24900992 NM_001290256 yes chr14 24896641 24901641 NM_001290257 yes chr16 89003945 89008945 NM_001290330 yes chr11 1853040 1858040 NM_001290332 yes chr1 225995336 226000336 NM_001291163 yes chr1 41825103 41830103 NM_001291281 yes chr19 14181321 14186321 NM_001291291 yes chr19 47985021 47990021 NM_001291296 yes chr19 15527432 15532432 NM_001291478 yes chr1 113006663 113011663 NM_001291880 yes chr5 176511373 176516373 NM_001291980 yes chr6 134493534 134498534 NM_001291995 yes chr2 170678941 170683941 NM_001293186 yes chr1 32227164 32232164 NM_001294335 yes chr5 41922854 41927854 NM_001297437 yes chr19 7966165 7971165 NM_001297555 yes chr19 45906967 45911967 NM_001297590 yes chr1 27690857 27695857 NM_001297609 yes chr1 205222829 205227829 NM_001297613 yes chr11 2419218 2424218 NM_001297658 yes chr11 2419748 2424748 NM_001297659 yes chr1 51761133 51766133 NM_001297666 yes chr1 183602682 183607682 NM_001297669 yes chr1 10000481 10005481 NM_001297778 yes chr1 10000986 10005986 NM_001297779 yes chr1 45137780 45142780 NM_001300746 yes chr12 49502183 49507183 NM_001300750 yes chr1 172419533 172424533 NM_001300760 yes chr11 62377737 62382737 NM_001300793 yes chr19 1266765 1271765 NM_001300815 yes chr12 53736077 53741077 NM_001300837 yes chr1 150252443 150257443 NM_001300838 yes chr1 150252632 150257632 NM_001300841 yes chr12 69750990 69755990 NM_001300950 yes chr5 140016512 140021512 NM_001300980 yes chr7 44833735 44838735 NM_001300981 yes chr15 75333110 75338110 NM_001301104 yes chr12 108952739 108957739 NM_001301140 yes chr15 45470905 45475905 NM_001301171 yes chr15 40613757 40618757 NM_001301268 yes chr2 159310765 159315765 NM_001301684 yes chr17 38714765 38719765 NM_001301716 yes chr17 38714146 38719146 NM_001301718 yes chr16 31467675 31472675 NM_001301820 yes chr12 7050165 7055165 NM_001301834 yes chr12 7050101 7055101 NM_001301836 yes chr12 7050480 7055480 NM_001301837 yes chr20 39966993 39971993 NM_001301860 yes chr19 11544103 11549103 NM_001302453 yes chr19 11543480 11548480 NM_001302454 yes chr7 100491254 100496254 NM_001302622 yes chr19 45406778 45411778 NM_001302689 yes chr19 45407158 45412158 NM_001302690 yes chr11 65335430 65340430 NM_001303024 yes chr1 38153767 38158767 NM_001303030 yes chr1 145522391 145527391 NM_001303040 yes chr7 100074402 100079402 NM_001303043 yes chr2 27235122 27240122 NM_001303050 yes chr1 202895272 202900272 NM_001303051 yes chr1 156695731 156700731 NM_001303095 yes chr2 220405885 220410885 NM_001303098 yes chr1 202893900 202898900 NM_001303109 yes chr12 56509504 56514504 NM_001303124 yes chr12 56509844 56514844 NM_001303125 yes chr3 150123622 150128622 NM_001303264 yes chr17 73006259 73011259 NM_001303265 yes chr17 7618578 7623578 NM_001303270 yes chr17 65711449 65716449 NM_001303272 yes chr1 78442389 78447389 NM_001303433 yes chr7 1081709 1086709 NM_001303473 yes chr19 39830599 39835599 NM_001303614 yes chrX 153624213 153629213 NM_001303624 yes chrX 153624338 153629338 NM_001303626 yes chr1 156021116 156026116 NM_001304342 yes chr2 113339514 113344514 NM_001304353 yes chr2 113339949 113344949 NM_001304354 yes chr6 44184742 44189742 NM_001304462 yes chr1 203828213 203833213 NM_001304464 yes chr16 19894456 19899456 NM_001304771 yes chr16 788538 793538 NM_001304799 yes chr12 49738200 49743200 NM_001304944 yes chr6 41886385 41891385 NM_001305455 yes chr6 41886181 41891181 NM_001305456 yes chr17 56492443 56497443 NM_001305544 yes chr11 64320573 64325573 NM_001307985 yes chr18 44134232 44139232 NM_001308013 yes chr3 196666811 196671811 NM_001308036 yes chr3 195617155 195622155 NM_001308046 yes chr5 133857566 133862566 NM_001308143 yes chr5 157095988 157100988 NM_001308165 yes chr8 145908697 145913697 NM_001308208 yes chr1 78146612 78151612 NM_001308237 yes chr1 94372654 94377654 NM_001308253 yes chr18 43301588 43306588 NM_001308278 yes chr16 57699657 57704657 NM_001308360 yes chr19 35998912 36003912 NM_001308380 yes chr19 50977503 50982503 NM_001308429 yes chr21 44343208 44348208 NM_001308491 yes chr1 202308594 202313594 NM_001310326 yes chr16 31103820 31108820 NM_001311311 yes chr5 76009368 76014368 NM_001311313 yes chr1 113613292 113618292 NM_001312686 yes chr2 178127359 178132359 NM_001313902 yes chr6 36722686 36727686 NM_001314018 yes chr10 6242340 6247340 NM_001314063 yes chr21 47742302 47747302 NM_001315529 yes chr3 52006542 52011542 NM_001316331 yes chr1 54409481 54414481 NM_001316935 yes chr1 10000985 10005985 NM_001316973 yes chr6 150282610 150287610 NM_001317089 yes chr1 78443368 78448368 NM_001317099 yes chr1 78442342 78447342 NM_001317100 yes chr1 36346296 36351296 NM_001317122 yes chr3 48591856 48596856 NM_001317134 yes chr3 48596113 48601113 NM_001317136 yes chr19 857159 862159 NM_001317335 yes chr6 13613059 13618059 NM_001317724 yes chr9 116160900 116165900 NM_001317745 yes chr8 98878749 98883749 NM_001317748 yes chr8 71518194 71523194 NM_001317804 yes chr8 71517426 71522426 NM_001317805 yes chr8 23383808 23388808 NM_001317812 yes chr8 124778179 124783179 NM_001317917 yes chr20 30309302 30314302 NM_001317920 yes chr9 131462283 131467283 NM_001317926 yes chr2 46767196 46772196 NM_001318063 yes chr9 33445131 33450131 NM_001318144 yes chr9 124919598 124924598 NM_001318195 yes chr7 26330165 26335165 NM_001318199 yes chr4 40056024 40061024 NM_001318359 yes chrX 107016718 107021718 NM_001318468 yes chr6 56817273 56822273 NM_001318539 yes chr18 710164 715164 NM_001318759 yes chr11 72522951 72527951 NM_001318766 yes chr11 66185975 66190975 NM_001318804 yes chr14 24096824 24101824 NM_001318835 yes chr16 4362262 4367262 NM_001318918 yes chr6 2997892 3002892 NM_001318940 yes chr22 30659306 30664306 NM_001319108 yes chr1 12674288 12679288 NM_001319225 yes chr10 102818499 102823499 NM_001319303 yes chr1 159748293 159753293 NM_001319658 yes chr1 159748236 159753236 NM_001319659 yes chr12 6980021 6985021 NM_001319670 yes chr17 7755884 7760884 NM_001319941 yes chr17 28658577 28663577 NM_001319942 yes chr1 114445415 114450415 NM_001319946 yes chr1 114444741 114449741 NM_001319947 yes chr1 158967602 158972602 NM_001320010 yes chr3 47514949 47519949 NM_001320044 yes chr1 160065979 160070979 NM_001320247 yes chr1 35322917 35327917 NM_001320261 yes chr1 76079194 76084194 NM_001320283 yes chr12 53898922 53903922 NM_001320296 yes chr17 7255955 7260955 NM_001320435 yes chr9 77639567 77644567 NM_001320497 yes chr3 138308298 138313298 NM_001320600 yes chr21 44525216 44530216 NM_001320646 yes chr1 101699805 101704805 NM_001320730 yes chr1 45954340 45959340 NM_001320800 yes chr1 212001080 212006080 NM_001320808 yes chr10 81317127 81322127 NM_001320814 yes chr1 38453282 38458282 NM_001320830 yes chr19 35643125 35648125 NM_001320912 yes chr17 7758709 7763709 NM_001320924 yes chr15 45692015 45697015 NM_001321015 yes chr19 47536087 47541087 NM_001321086 yes chr19 35757381 35762381 NM_001321150 yes chr19 1064997 1069997 NM_001321232 yes chr11 108090865 108095865 NM_001321307 yes chr17 43207467 43212467 NM_001321352 yes chr17 1529680 1534680 NM_001321364 yes chr12 124066578 124071578 NM_001321445 yes chr2 201372317 201377317 NM_001321547 yes chr15 89087106 89092106 NM_001321596 yes chr8 71313973 71318973 NM_001321703 yes chr8 71313127 71318127 NM_001321707 yes chr8 71313575 71318575 NM_001321711 yes chr8 71312990 71317990 NM_001321712 yes chr1 154931447 154936447 NM_001321726 yes chr1 25662245 25667245 NM_001321772 yes chr2 55275453 55280453 NM_001321859 yes chr2 55275234 55280234 NM_001321860 yes chr2 55274327 55279327 NM_001321861 yes chr2 55273302 55278302 NM_001321862 yes chr2 55271522 55276522 NM_001321863 yes chr20 30307855 30312855 NM_001322242 yes chr1 109630863 109635863 NM_001322248 yes chr14 60629711 60634711 NM_001322281 yes chr1 249117065 249122065 NM_001322462 yes chr1 249117984 249122984 NM_001322464 yes chr20 33541330 33546330 NM_001322494 yes chr20 33540938 33545938 NM_001322495 yes chr1 24737477 24742477 NM_001322854 yes chr1 24739740 24744740 NM_001322855 yes chr1 24740015 24745015 NM_001322857 yes chr1 154912626 154917626 NM_001323012 yes chr15 91475350 91480350 NM_001323619 yes chr15 91475616 91480616 NM_001323620 yes chr15 45692019 45697019 NM_001323640 yes chr1 151040554 151045554 NM_001323906 yes chr1 227913719 227918719 NM_001323930 yes chr1 227913369 227918369 NM_001323933 yes chr17 72560982 72565982 NM_001324073 yes chr16 835883 840883 NM_001324086 yes chr16 68025694 68030694 NM_001324159 yes chr16 75654721 75659721 NM_001324444 yes chr1 175710252 175715252 NM_001328635 yes chr1 203828478 203833478 NM_001328637 yes chr1 43994031 43999031 NM_001329139 yes chr7 135240162 135245162 NM_001329434 yes chr8 145700865 145705865 NM_001329442 yes chr8 145701234 145706234 NM_001329444 yes chr7 86847403 86852403 NM_001329472 yes chr7 86847168 86852168 NM_001329475 yes chr17 7758545 7763545 NM_001330110 yes chr17 37884316 37889316 NM_001330206 yes chr12 54066703 54071703 NM_001330269 yes chr1 1281992 1286992 NM_001330311 yes chr17 46657484 46662484 NM_001330322 yes chr12 6872983 6877983 NM_001330333 yes chr1 32714340 32719340 NM_001330468 yes chr17 72730199 72735199 NM_001330471 yes chr19 38823927 38828927 NM_001330496 yes chr17 73849393 73854393 NM_001330499 yes chr8 96034703 96039703 NM_001330582 yes chr8 145685734 145690734 NM_001330618 yes chr1 151168520 151173520 NM_001330689 yes chr1 151224676 151229676 NM_001330692 yes chr9 100682352 100687352 NM_001330725 yes chr1 159793979 159798979 NM_001330741 yes chr1 205223825 205228825 NM_001331034 yes chr22 21984586 21989586 NM_001331066 yes chr17 72361145 72366145 NM_001331076 yes chr13 27842814 27847814 NM_001331126 yes chr12 56322446 56327446 NM_001345 yes chr11 62310738 62315738 NM_001346445 yes chr11 62310659 62315659 NM_001346446 yes chr17 66199770 66204770 NM_001346471 yes chr6 109700442 109705442 NM_001346500 yes chr17 1930906 1935906 NM_001346574 yes chr19 39733217 39738217 NM_001346937 yes chr11 118970285 118975285 NM_001382 yes chr19 11589303 11594303 NM_001420 yes chr6 52857678 52862678 NM_001512 yes chr1 32080801 32085801 NM_001525 yes chr17 38597176 38602176 NM_001552 yes chr1 86043944 86048944 NM_001554 yes chr11 67031405 67036405 NM_001619 yes chr3 186328350 186333350 NM_001622 yes chr1 161190918 161195918 NM_001643 yes chr11 3859713 3864713 NM_001665 yes chr11 117692959 117697959 NM_001680 yes chr3 187461013 187466013 NM_001706 yes chr12 92537173 92542173 NM_001731 yes chr12 7242543 7247543 NM_001733 yes chr21 36419095 36424095 NM_001754 yes chr14 23586320 23591320 NM_001805 yes chr20 3764837 3769837 NM_001810 yes chr5 175841070 175846070 NM_001834 yes chr1 40780439 40785439 NM_001852 yes chr1 156672959 156677959 NM_001878 yes chr17 61521222 61526222 NM_001915 yes chr5 139723688 139728688 NM_001945 yes chr6 20399637 20404637 NM_001949 yes chr1 205599500 205604500 NM_001973 yes chr12 56471309 56476309 NM_001982 yes chr15 91425188 91430188 NM_002005 yes chr1 171280822 171285822 NM_002022 yes chr1 27996224 28001224 NM_002038 yes chr17 73399289 73404289 NM_002086 yes chr17 42419954 42424954 NM_002087 yes chr7 26237913 26242913 NM_002137 yes chr1 33349598 33354598 NM_002143 yes chr17 46668603 46673603 NM_002147 yes chr12 53488936 53493936 NM_002178 yes chr1 59247285 59252285 NM_002228 yes chr13 46753959 46758959 NM_002298 yes chr12 120804483 120809483 NM_002442 yes chr1 155175990 155180990 NM_002455 yes chr8 128745815 128750815 NM_002467 yes chr1 203052666 203057666 NM_002479 yes chr5 141255475 141260475 NM_002587 yes chr20 5104768 5109768 NM_002592 yes chr7 100197382 100202382 NM_002593 yes chr1 172410730 172415730 NM_002642 yes chr20 39763661 39768661 NM_002660 yes chr15 74284514 74289514 NM_002675 yes chr17 37822206 37827206 NM_002686 yes chr6 105848499 105853499 NM_002726 yes chr19 14226059 14231059 NM_002730 yes chr19 14541666 14546666 NM_002741 yes chr19 11543966 11548966 NM_002743 yes chr1 151224697 151229697 NM_002810 yes chr20 25226206 25231206 NM_002862 yes chr19 18312374 18317374 NM_002866 yes chr1 153956353 153961353 NM_002870 yes chr1 182571048 182576048 NM_002928 yes chr1 153327830 153332830 NM_002965 yes chr1 151964214 151969214 NM_002966 yes chrX 23798775 23803775 NM_002970 yes chr19 51224105 51229105 NM_002975 yes chr1 168543211 168548211 NM_002995 yes chr7 37954025 37959025 NM_003014 yes chr9 38066710 38071710 NM_003028 yes chr2 65213995 65218995 NM_003038 yes chr7 150754157 150759157 NM_003040 yes chr1 27479121 27484121 NM_003047 yes chr12 50476483 50481483 NM_003076 yes chr9 139290389 139295389 NM_003086 yes chr6 34722371 34727371 NM_003093 yes chr1 168510735 168515735 NM_003175 yes chr12 50132793 50137793 NM_003217 yes chr11 1857733 1862733 NM_003282 yes chr1 186341957 186346957 NM_003292 yes chr11 2948150 2953150 NM_003311 yes chr17 40167215 40172215 NM_003315 yes chrX 47050701 47055701 NM_003334 yes chr3 48644598 48649598 NM_003365 yes chr10 17267758 17272758 NM_003380 yes chr12 49363141 49368141 NM_003394 yes chr1 228133176 228138176 NM_003395 yes chr11 107876908 107881908 NM_003478 yes chr16 87900605 87905605 NM_003486 yes chr6 27858463 27863463 NM_003514 yes chr1 149811818 149816818 NM_003516 yes chr1 149820128 149825128 NM_003516_2 yes chr1 149856025 149861025 NM_003517 yes chr6 27858703 27863703 NM_003527 yes chr1 149855732 149860732 NM_003528 yes chr1 149830225 149835225 NM_003548 yes chr1 149801721 149806721 NM_003548_2 yes chr1 150951999 150956999 NM_003568 yes chr15 65501340 65506340 NM_003613 yes chr17 40832132 40837132 NM_003632 yes chr17 77810713 77815713 NM_003655 yes chr3 5018597 5023597 NM_003670 yes chr5 172754006 172759006 NM_003714 yes chr9 140080554 140085554 NM_003731 yes chr6 39194751 39199751 NM_003740 yes chr16 11347539 11352539 NM_003745 yes chr12 120905058 120910058 NM_003769 yes chr17 39643616 39648616 NM_003771 yes chr3 50357781 50362781 NM_003773 yes chr12 89916083 89921083 NM_003774 yes chr20 5104907 5109907 NM_003818 yes chr19 48016015 48021015 NM_003827 yes chr1 167520556 167525556 NM_003851 yes chr12 93961098 93966098 NM_003877 yes chr5 141013923 141018923 NM_003883 yes chr10 72573204 72578204 NM_003901 yes chr8 145699218 145704218 NM_003923 yes chr17 76353660 76358660 NM_003955 yes chr9 35789906 35794906 NM_003995 yes chr6 41886465 41891465 NM_004053 yes chr1 45263397 45268397 NM_004073 yes chr12 56691675 56696675 NM_004077 yes chrX 106957791 106962791 NM_004089 yes chr8 37885520 37890520 NM_004095 yes chr20 30430920 30435920 NM_004118 yes chr11 65653510 65658510 NM_004214 yes chr11 57332680 57337680 NM_004223 yes chr12 121085810 121090810 NM_004276 yes chr1 9292363 9297363 NM_004285 yes chr1 25254270 25259270 NM_004350 yes chr12 120873393 120878393 NM_004373 yes chr11 118659472 118664472 NM_004397 yes chr5 172195703 172200703 NM_004417 yes chr8 22548315 22553315 NM_004430 yes chr9 131578279 131583279 NM_004435 yes chr17 65819280 65824280 NM_004459 yes chr1 47899189 47904189 NM_004474 yes chr19 46365018 46370018 NM_004497 yes chr6 44231025 44236025 NM_004556 yes chr14 68064517 68069517 NM_004569 yes chr14 24738333 24743333 NM_004581 yes chr11 67156923 67161923 NM_004584 yes chr19 46193074 46198074 NM_004597 yes chr16 30100705 30105705 NM_004608 yes chr11 75915074 75920074 NM_004626 yes chr12 123754187 123759187 NM_004642 yes chr16 67512589 67517589 NM_004691 yes chr3 51973457 51978457 NM_004704 yes chr15 55698074 55703074 NM_004748 yes chr1 12675237 12680237 NM_004753 yes chr1 52605266 52610266 NM_004799 yes chr12 49243457 49248457 NM_004818 yes chr19 46364048 46369048 NM_004819 yes chr19 14140052 14145052 NM_004843 yes chr19 50866587 50871587 NM_004851 yes chr17 7515715 7520715 NM_004860 yes chr19 18494270 18499270 NM_004864 yes chr10 43902156 43907156 NM_004966 yes chr1 160048860 160053860 NM_004983 yes chr19 35627863 35632863 NM_005031 yes chr1 151801848 151806848 NM_005060 yes chr1 145505057 145510057 NM_005105 yes chr3 38385751 38390751 NM_005108 yes chr3 38204526 38209526 NM_005109 yes chr17 60140143 60145143 NM_005121 yes chr22 50961534 50966534 NM_005138 yes chr1 113255450 113260450 NM_005167 yes chr12 54067609 54072609 NM_005176 yes chr11 64946804 64951804 NM_005186 yes chr11 119074486 119079486 NM_005188 yes chr17 77749477 77754477 NM_005189 yes chr17 1357061 1362061 NM_005206 yes chr22 21269214 21274214 NM_005207 yes chr1 155033713 155038713 NM_005227 yes chr1 92949933 92954933 NM_005263 yes chr1 27716648 27721648 NM_005281 yes chr19 35839945 35844945 NM_005303 yes chr17 73773516 73778516 NM_005324 yes chr19 16219990 16224990 NM_005370 yes chr1 146641668 146646668 NM_005399 yes chr12 49369736 49374736 NM_005430 yes chr12 6859582 6864582 NM_005439 yes chr5 141390120 141395120 NM_005471 yes chr12 111841252 111846252 NM_005475 yes chr19 6765023 6770023 NM_005490 yes chr11 65623304 65628304 NM_005507 yes chr1 26796402 26801402 NM_005517 yes chr3 193851431 193856431 NM_005524 yes chr1 152007011 152012011 NM_005620 yes chr1 29506137 29511137 NM_005626 yes chr2 231787441 231792441 NM_005683 yes chr11 65360967 65365967 NM_005714 yes chr10 104260012 104265012 NM_005736 yes chr1 47653271 47658271 NM_005764 yes chr12 7123342 7128342 NM_005768 yes chr12 56613215 56618215 NM_005785 yes chr3 183964259 183969259 NM_005787 yes chr17 39842627 39847627 NM_005801 yes chr7 100301176 100306176 NM_005837 yes chr11 67273699 67278699 NM_005851 yes chr3 136468745 136473745 NM_005862 yes chr2 220297200 220302200 NM_005876 yes chr17 43296792 43301792 NM_005892 yes chr2 209117367 209122367 NM_005896 yes chr2 85763601 85768601 NM_005911 yes chr1 156468134 156473134 NM_005920 yes chr3 196754187 196759187 NM_005929 yes chr19 6277459 6282459 NM_005934 yes chr1 38322792 38327792 NM_005955 yes chr1 153535806 153540806 NM_005978 yes chr1 153598215 153603215 NM_005979 yes chr22 19163876 19168876 NM_005984 yes chr20 48597013 48602013 NM_005985 yes chr22 19741726 19746726 NM_005992 yes chr21 47741536 47746536 NM_006031 yes chr12 49522804 49527804 NM_006082 yes chr14 24627922 24632922 NM_006084 yes chr12 48149744 48154744 NM_006105 yes chr1 27187133 27192133 NM_006142 yes chr12 54687063 54692063 NM_006163 yes chr2 201825924 201830924 NM_006190 yes chr3 178863811 178868811 NM_006218 yes chr12 49686409 49691409 NM_006262 yes chr1 153598373 153603373 NM_006271 yes chr2 112653691 112658691 NM_006343 yes chr9 100742989 100747989 NM_006401 yes chr19 42461028 42466028 NM_006423 yes chr17 39965951 39970951 NM_006455 yes chr17 43222184 43227184 NM_006460 yes chr1 110088686 110093686 NM_006496 yes chr17 1955893 1960893 NM_006497 yes chr20 52197136 52202136 NM_006526 yes chr14 50996876 51001876 NM_006575 yes chr1 156644689 156649689 NM_006617 yes chr11 72502250 72507250 NM_006645 yes chr6 41745143 41750143 NM_006653 yes chr19 45905812 45910812 NM_006663 yes chr6 34357957 34362957 NM_006703 yes chr11 118824508 118829508 NM_006760 yes chr1 87791651 87796651 NM_006769 yes chr11 65079789 65084789 NM_006779 yes chr11 64643740 64648740 NM_006795 yes chr19 48892310 48897310 NM_006801 yes chr5 137087539 137092539 NM_006805 yes chr12 124066576 124071576 NM_006815 yes chr1 151029651 151034651 NM_006818 yes chr1 79083567 79088567 NM_006820 yes chr19 1602983 1607983 NM_006830 yes chr11 65655375 65660375 NM_006848 yes chr14 90860827 90865827 NM_006888 yes chr12 8086392 8091392 NM_006931 yes chr10 103540670 103545670 NM_006993 yes chr11 130296388 130301388 NM_007037 yes chr1 145713139 145718139 NM_007053 yes chr6 35433678 35438678 NM_007104 yes chr6 41700298 41705298 NM_007162 yes chr1 112295690 112300690 NM_007204 yes chr12 57608078 57613078 NM_007224 yes chr17 47650798 47655798 NM_007225 yes chr1 109286785 109291785 NM_007269 yes chr7 26238331 26243331 NM_007276 yes chr22 19947570 19952570 NM_007310 yes chr1 161733534 161738534 NM_007348 yes chr8 145548082 145553082 NM_012079 yes chr1 150227638 150232638 NM_012113 yes chr17 71305643 71310643 NM_012121 yes chr15 43474966 43479966 NM_012142 yes chr19 46140175 46145175 NM_012155 yes chr5 41922856 41927856 NM_012176 yes chr12 120737624 120742624 NM_012240 yes chr6 13572359 13577359 NM_012241 yes chr16 30454796 30459796 NM_012248 yes chr2 46767367 46772367 NM_012249 yes chr6 52439362 52444362 NM_012288 yes chr19 1064665 1069665 NM_012292 yes chr1 32571144 32576144 NM_012316 yes chr10 28963924 28968924 NM_012342 yes chr1 161085366 161090366 NM_012394 yes chr7 44885225 44890225 NM_012412 yes chr17 65370897 65375897 NM_012417 yes chr6 74361237 74366237 NM_012434 yes chr17 73849001 73854001 NM_012478 yes chr11 67138706 67143706 NM_013246 yes chr8 145632233 145637233 NM_013291 yes chr12 110903732 110908732 NM_013300 yes chr16 22215092 22220092 NM_013302 yes chr11 64792380 64797380 NM_013306 yes chr3 50646762 50651762 NM_013324 yes chr17 897633 902633 NM_013337 yes chr1 151146047 151151047 NM_013353 yes chr9 140080557 140085557 NM_013366 yes chr6 26594671 26599671 NM_013375 yes chr6 41746000 41751000 NM_013397 yes chr11 61582029 61587029 NM_013402 yes chr8 145667312 145672312 NM_013432 yes chr19 16293712 16298712 NM_014077 yes chr7 73621587 73626587 NM_014146 yes chr11 64882710 64887710 NM_014205 yes chr1 156826212 156831212 NM_014215 yes chr3 45633823 45638823 NM_014240 yes chr12 6935038 6940038 NM_014262 yes chr1 25662289 25667289 NM_014313 yes chr12 109122826 109127826 NM_014325 yes chr15 64336021 64341021 NM_014326 yes chr11 35637235 35642235 NM_014344 yes chr1 174966392 174971392 NM_014412 yes chr19 47731951 47736951 NM_014417 yes chr5 148756338 148761338 NM_014443 yes chr6 47275183 47280183 NM_014452 yes chr11 2441775 2446775 NM_014555 yes chr1 40102848 40107848 NM_014571 yes chr17 36505507 36510507 NM_014598 yes chr19 48214101 48219101 NM_014601 yes chr5 175872856 175877856 NM_014613 yes chr1 153506217 153511217 NM_014624 yes chr22 22304750 22309750 NM_014634 yes chr2 219573068 219578068 NM_014640 yes chr1 44409978 44414978 NM_014652 yes chr1 31378980 31383980 NM_014654 yes chr19 47850038 47855038 NM_014681 yes chr3 183964945 183969945 NM_014693 yes chr12 108952665 108957665 NM_014706 yes chr19 36206421 36211421 NM_014727 yes chr11 73017163 73022163 NM_014786 yes chr11 72850643 72855643 NM_014824 yes chr12 57397797 57402797 NM_014830 yes chr1 153916654 153921654 NM_014856 yes chr1 205194538 205199538 NM_014858 yes chr1 212001614 212006614 NM_014873 yes chr10 94048375 94053375 NM_014912 yes chr6 52924100 52929100 NM_014920 yes chr19 12946759 12951759 NM_014975 yes chr7 129707849 129712849 NM_014997 yes chr1 39872676 39877676 NM_015038 yes chr1 10268264 10273264 NM_015074 yes chr11 118474713 118479713 NM_015157 yes chr8 67338763 67343763 NM_015169 yes chr1 171452166 171457166 NM_015172 yes chr11 111470615 111475615 NM_015191 yes chr10 101767210 101772210 NM_015221 yes chr5 133859298 133864298 NM_015288 yes chr22 32338848 32343848 NM_015372 yes chr9 139938176 139943176 NM_015392 yes chr4 113556112 113561112 NM_015454 yes chr10 102277095 102282095 NM_015490 yes chr15 75491721 75496721 NM_015492 yes chr2 171782536 171787536 NM_015530 yes chr1 78145843 78150843 NM_015534 yes chr1 156568779 156573779 NM_015590 yes chr20 33678118 33683118 NM_015638 yes chr19 46386928 46391928 NM_015649 yes chr19 2473623 2478623 NM_015675 yes chr19 48246293 48251293 NM_015710 yes chr7 100491041 100496041 NM_015831 yes chr1 26493888 26498888 NM_015871 yes chr12 121016697 121021697 NM_015918 yes chr17 7195376 7200376 NM_015982 yes chr8 71578947 71583947 NM_016027 yes chr11 66203819 66208819 NM_016050 yes chr7 100885871 100890871 NM_016068 yes chr19 12777965 12782965 NM_016145 yes chr16 19893733 19898733 NM_016235 yes chr1 202933904 202938904 NM_016243 yes chr19 49337434 49342434 NM_016246 yes chr6 17598018 17603018 NM_016255 yes chr1 29060636 29065636 NM_016258 yes chr10 76856748 76861748 NM_016364 yes chr11 10560274 10565274 NM_016422 yes chr2 97521256 97526256 NM_016466 yes chr5 141336127 141341127 NM_016580 yes chr17 40831345 40836345 NM_016602 yes chr12 54376446 54381446 NM_017409 yes chr11 64609541 64614541 NM_017525 yes chr3 122397172 122402172 NM_017554 yes chr17 33446131 33451131 NM_017559 yes chr19 2048743 2053743 NM_017572 yes chr1 154528620 154533620 NM_017582 yes chr10 104675575 104680575 NM_017649 yes chr2 97533235 97538235 NM_017789 yes chr10 94048420 94053420 NM_017824 yes chr1 36551953 36556953 NM_017825 yes chr1 27111954 27116954 NM_017837 yes chr8 95833018 95838018 NM_017864 yes chr10 102293141 102298141 NM_017902 yes chr14 55735521 55740521 NM_017943 yes chr9 77565302 77570302 NM_017998 yes chr5 150078169 150083169 NM_018047 yes chr7 4920835 4925835 NM_018059 yes chr1 201795788 201800788 NM_018085 yes chr3 72895098 72900098 NM_018130 yes chr19 14244940 14249940 NM_018154 yes chr1 205323628 205328628 NM_018203 yes chr1 65208278 65213278 NM_018211 yes chr8 37704931 37709931 NM_018310 yes chr17 48553661 48558661 NM_018346 yes chr3 194390706 194395706 NM_018385 yes chr4 113555651 113560651 NM_018392 yes chr12 10824391 10829391 NM_018423 yes chr1 11721650 11726650 NM_018438 yes chr17 48472414 48477414 NM_018509 yes chr1 207221922 207226922 NM_018566 yes chr15 34632862 34637862 NM_018648 yes chr15 90292040 90297040 NM_018670 yes chr17 71186673 71191673 NM_018714 yes chr1 207036654 207041654 NM_018724 yes chr17 46679834 46684834 NM_018952 yes chr12 54424332 54429332 NM_018953 yes chr12 122229094 122234094 NM_019034 yes chr10 74031177 74036177 NM_019058 yes chr15 41219031 41224031 NM_019074 yes chr19 42744236 42749236 NM_019884 yes chr1 109630903 109635903 NM_020141 yes chr12 57631975 57636975 NM_020142 yes chr19 49942308 49947308 NM_020309 yes chr22 38052237 38057237 NM_020315 yes chr4 71551696 71556696 NM_020368 yes chr17 37305402 37310402 NM_020405 yes chr20 42813718 42818718 NM_020433 yes chr1 24739745 24744745 NM_020448 yes chr1 26124167 26129167 NM_020451 yes chr1 154297776 154302776 NM_020452 yes chr16 67873713 67878713 NM_020457 yes chr14 35871460 35876460 NM_020529 yes chr20 24970925 24975925 NM_020531 yes chr17 73509164 73514164 NM_020753 yes chr1 11536795 11541795 NM_020780 yes chr15 74420243 74425243 NM_020851 yes chr15 41184128 41189128 NM_020857 yes chr17 7618172 7623172 NM_020877 yes chr20 33541138 33546138 NM_020884 yes chr1 33205012 33210012 NM_020888 yes chr12 125397087 125402087 NM_021009 yes chr12 56549545 56554545 NM_021019 yes chr12 49486102 49491102 NM_021044 yes chr11 1966002 1971002 NM_021134 yes chr7 92074262 92079262 NM_021167 yes chr19 38823943 38828943 NM_021185 yes chr2 176962030 176967030 NM_021193 yes chr1 211749599 211754599 NM_021194 yes chr10 75399015 75404015 NM_021245 yes chr20 44934637 44939637 NM_021248 yes chr17 8019360 8024360 NM_021628 yes chr2 12854498 12859498 NM_021643 yes chr19 35627193 35632193 NM_021902 yes chr6 35417638 35422638 NM_021922 yes chr17 39966462 39971462 NM_021939 yes chr19 35631654 35636654 NM_022006 yes chr22 21994042 21999042 NM_022044 yes chr6 35263095 35268095 NM_022047 yes chr11 6702132 6707132 NM_022061 yes chr11 125032059 125037059 NM_022062 yes chr16 836122 841122 NM_022092 yes chr1 40234520 40239520 NM_022120 yes chr6 138426160 138431160 NM_022121 yes chr1 179048612 179053612 NM_022371 yes chr17 880498 885498 NM_022463 yes chr3 178787156 178792156 NM_022470 yes chr14 23524247 23529247 NM_022478 yes chr2 74707700 74712700 NM_022492 yes chr7 100284370 100289370 NM_022574 yes chr12 54400390 54405390 NM_022658 yes chr22 19129690 19134690 NM_022719 yes chr11 62356609 62361609 NM_022830 yes chr19 39901250 39906250 NM_022835 yes chr5 176514051 176519051 NM_022963 yes chr1 32799340 32804340 NM_023009 yes chr1 53161538 53166538 NM_023077 yes chr19 17413977 17418977 NM_023937 yes chr16 762673 767673 NM_024042 yes chr12 56615625 56620625 NM_024068 yes chr7 99814371 99819371 NM_024070 yes chr16 30438873 30443873 NM_024096 yes chr1 43230416 43235416 NM_024097 yes chr19 981828 986828 NM_024100 yes chr12 102222145 102227145 NM_024312 yes chr7 86846814 86851814 NM_024315 yes chr2 232788613 232793613 NM_024409 yes chr18 43649750 43654750 NM_024430 yes chr16 29825028 29830028 NM_024516 yes chr19 17411782 17416782 NM_024527 yes chr1 193088588 193093588 NM_024529 yes chr2 220405987 220410987 NM_024536 yes chr1 151126605 151131605 NM_024575 yes chr17 78963141 78968141 NM_024591 yes chr8 9005720 9010720 NM_024607 yes chr1 38271365 38276365 NM_024640 yes chr16 30535410 30540410 NM_024671 yes chr1 220861128 220866128 NM_024709 yes chr17 40826548 40831548 NM_024927 yes chr11 65625372 65630372 NM_025128 yes chr3 196693242 196698242 NM_025163 yes chr9 35113408 35118408 NM_025182 yes chr8 126440063 126445063 NM_025195 yes chr4 7067437 7072437 NM_025196 yes chr3 52310159 52315159 NM_025222 yes chr12 12712948 12717948 NM_030640 yes chr1 249117654 249122654 NM_030645 yes chr19 46316105 46321105 NM_030785 yes chr11 75234099 75239099 NM_030792 yes chr20 30537383 30542383 NM_030815 yes chr15 73073626 73078626 NM_031284 yes chr1 26603713 26608713 NM_031286 yes chr3 46536985 46541985 NM_031440 yes chr11 63971652 63976652 NM_031471 yes chr19 54413491 54418491 NM_031896 yes chr14 94593457 94598457 NM_032036 yes chr1 202429371 202434371 NM_032103 yes chr16 67698159 67703159 NM_032140 yes chr5 125934107 125939107 NM_032177 yes chr11 64105190 64110190 NM_032251 yes chr1 27150701 27155701 NM_032283 yes chr1 245130671 245135671 NM_032328 yes chr19 12778017 12783017 NM_032332 yes chr12 56319197 56324197 NM_032345 yes chr17 46797382 46802382 NM_032391 yes chr15 45720227 45725227 NM_032413 yes chr10 98590212 98595212 NM_032440 yes chr17 73898681 73903681 NM_032478 yes chr1 40135210 40140210 NM_032526 yes chr2 27502797 27507797 NM_032546 yes chr19 914842 919842 NM_032551 yes chr14 21507885 21512885 NM_032572 yes chr16 4379725 4384725 NM_032575 yes chr7 23051270 23056270 NM_032581 yes chr17 48225378 48230378 NM_032595 yes chr1 32710318 32715318 NM_032648 yes chr8 145688349 145693349 NM_032687 yes chr22 20847670 20852670 NM_032775 yes chr2 74707857 74712857 NM_032779 yes chr12 122061955 122066955 NM_032790 yes chr6 150182980 150187980 NM_032832 yes chr17 27945941 27950941 NM_032854 yes chr15 85195074 85200074 NM_032856 yes chr11 73085213 73090213 NM_032871 yes chr12 53643376 53648376 NM_032889 yes chr16 57494051 57499051 NM_032940 yes chr3 196754186 196759186 NM_033316 yes chr17 28254558 28259558 NM_033389 yes chr11 109961587 109966587 NM_033390 yes chr9 37901850 37906850 NM_033412 yes chr15 40396139 40401139 NM_033503 yes chr9 100952456 100957456 NM_052820 yes chr22 37821005 37826005 NM_052906 yes chr22 42320321 42325321 NM_052945 yes chr8 11321776 11326776 NM_053279 yes chr2 219855627 219860627 NM_057093 yes chr1 55462117 55467117 NM_057176 yes chr8 95904982 95909982 NM_057749 yes chr1 32401488 32406488 NM_080391 yes chr11 78126368 78131368 NM_080491 yes chr12 7053240 7058240 NM_080548 yes chr5 133745098 133750098 NM_080656 yes chr20 631514 636514 NM_080725 yes chr20 49250955 49255955 NM_080829 yes chr1 160922089 160927089 NM_080878 yes chr7 37023371 37028371 NM_130442 yes chr17 38373074 38378074 NM_133264 yes chr5 176235060 176240060 NM_133369 yes chr17 43207400 43212400 NM_133373 yes chr1 198123608 198128608 NM_133494 yes chr15 41163987 41168987 NM_133639 yes chr13 21274982 21279982 NM_138284 yes chr1 154931758 154936758 NM_138300 yes chr3 183945717 183950717 NM_138345 yes chr15 45490873 45495873 NM_138356 yes chr8 145978470 145983470 NM_138367 yes chr11 65545562 65550562 NM_138368 yes chr14 20771653 20776653 NM_138376 yes chr8 145731919 145736919 NM_138431 yes chr19 18043405 18048405 NM_138442 yes chr14 24766166 24771166 NM_138452 yes chr17 27050730 27055730 NM_138463 yes chr1 75196340 75201340 NM_138467 yes chr8 145687918 145692918 NM_138496 yes chr6 126275361 126280361 NM_138571 yes chr19 997918 1002918 NM_138690 yes chr6 149885028 149890028 NM_138785 yes chr19 1809775 1814775 NM_138813 yes chr11 124629723 124634723 NM_138961 yes chr5 5137943 5142943 NM_139056 yes chr6 149864738 149869738 NM_139126 yes chr19 35623678 35628678 NM_139284 yes chr9 116204509 116209509 NM_144488 yes chr9 116353700 116358700 NM_144489 yes chr12 56658142 56663142 NM_144576 yes chr19 48820832 48825832 NM_144577 yes chr10 76993270 76998270 NM_144589 yes chr18 44234496 44239496 NM_144612 yes chr1 151040580 151045580 NM_144618 yes chr17 27227589 27232589 NM_144683 yes chr1 156887924 156892924 NM_144702 yes chr19 35643172 35648172 NM_144779 yes chr19 47161895 47166895 NM_145056 yes chr1 33935732 33940732 NM_145238 yes chr1 59009971 59014971 NM_145243 yes chr2 11884222 11889222 NM_145693 yes chr8 145689220 145694220 NM_145754 yes chr11 34377055 34382055 NM_145804 yes chr19 49173838 49178838 NM_145807 yes chr18 12656238 12661238 NM_147163 yes chr17 38518445 38523445 NM_152219 yes chr11 73084905 73089905 NM_152222 yes chr14 55736372 55741372 NM_152231 yes chr10 98477779 98482779 NM_152309 yes chr17 27918027 27923027 NM_152345 yes chr19 40021007 40026007 NM_152361 yes chr1 40994733 40999733 NM_152373 yes chr2 170333506 170338506 NM_152384 yes chr8 96034714 96039714 NM_152416 yes chr9 77640846 77645846 NM_152420 yes chr14 68166103 68171103 NM_152443 yes chr1 95580394 95585394 NM_152487 yes chr22 42093018 42098018 NM_152513 yes chr2 230931215 230936215 NM_152527 yes chr16 57123955 57128955 NM_152727 yes chr19 1235490 1240490 NM_152769 yes chr9 35094098 35099098 NM_152850 yes chr2 178974651 178979651 NM_152945 yes chr5 96476020 96481020 NM_153234 yes chr1 45250926 45255926 NM_153274 yes chrX 47047699 47052699 NM_153280 yes chr17 6944742 6949742 NM_153357 yes chr15 34656895 34661895 NM_153613 yes chr3 194404122 194409122 NM_153690 yes chr4 141442812 141447812 NM_153702 yes chr1 206969715 206974715 NM_153758 yes chr2 170681518 170686518 NM_172070 yes chr19 39756657 39761657 NM_172138 yes chr19 39733146 39738146 NM_172139 yes chr21 35733823 35738823 NM_172201 yes chr18 3451272 3456272 NM_173211 yes chr5 157168203 157173203 NM_173491 yes chr10 134256214 134261214 NM_173541 yes chr7 100079050 100084050 NM_173564 yes chr11 63681816 63686816 NM_173587 yes chr11 18745277 18750277 NM_173588 yes chr12 56649643 56654643 NM_173595 yes chr1 155143304 155148304 NM_173852 yes chr12 122105060 122110060 NM_173855 yes chr19 38891275 38896275 NM_174905 yes chr8 145595204 145600204 NM_174922 yes chr14 77605634 77610634 NM_174976 yes chr19 50977234 50982234 NM_175063 yes chr1 149856966 149861966 NM_175065 yes chr12 50098697 50103697 NM_175736 yes chr19 46269997 46274997 NM_175875 yes chr7 106299134 106304134 NM_175884 yes chr16 89005108 89010108 NM_175931 yes chr19 1246052 1251052 NM_177401 yes chr19 46192750 46197750 NM_177542 yes chr20 33458161 33463161 NM_178026 yes chr17 72917870 72922870 NM_178160 yes chr17 27053332 27058332 NM_178170 yes chr1 152746348 152751348 NM_178354 yes chr1 152593079 152598079 NM_178431 yes chr9 139962528 139967528 NM_178448 yes chr17 1926139 1931139 NM_178568 yes chr11 57225510 57230510 NM_178570 yes chr5 67581752 67586752 NM_181524 yes chr7 101458382 101463382 NM_181552 yes chr10 35413885 35418885 NM_181571 yes chr12 52398229 52403229 NM_181711 yes chr17 6923869 6928869 NM_181844 yes chr9 127536937 127541937 NM_182487 yes chr2 11270679 11275679 NM_182500 yes chr15 55579513 55584513 NM_183235 yes chr14 74224501 74229501 NM_194278 yes chr8 125382440 125387440 NM_194291 yes chr3 39231577 39236577 NM_194293 yes chr22 39926360 39931360 NM_194326 yes chr12 56613253 56618253 NM_194359 yes chr18 60380172 60385172 NM_194449 yes chrX 107016517 107021517 NM_198057 yes chr1 85722855 85727855 NM_198077 yes chr11 57333303 57338303 NM_198183 yes chr12 121016701 121021701 NM_198202 yes chr16 67916281 67921281 NM_198443 yes chr1 204180720 204185720 NM_198447 yes chr6 42844854 42849854 NM_198486 yes chr8 86155216 86160216 NM_198584 yes chr1 112295919 112300919 NM_198926 yes chr9 140116587 140121587 NM_199001 yes chr1 46803349 46808349 NM_199044 yes chr5 37833093 37838093 NM_199231 yes chr12 56323312 56328312 NM_201444 yes chr2 85643554 85648554 NM_201594 yes chr18 710017 715017 NM_202758 yes chr9 131938040 131943040 NM_203434 yes chr1 183619948 183624948 NM_203454 yes chr17 73399290 73404290 NM_203506 yes chr13 27841964 27846964 NM_206827 yes chr19 7988551 7993551 NM_206833 yes chr1 151412181 151417181 NM_207171 yes chr17 73510109 73515109 NM_207346 yes chr15 40630668 40635668 NM_207380 yes chr19 14222500 14227500 NM_207518 yes chr2 55273826 55278826 NM_207521 yes chrX 153626122 153631122 NR_000011 yes chr19 17970897 17975897 NR_000012 yes chr7 98868424 98873424 NR_002147 yes chr1 155194825 155199825 NR_002188 yes chr20 52489748 52494748 NR_002189 yes chr11 2016605 2021605 NR_002196 yes chr8 146217751 146222751 NR_002807 yes chr12 7074269 7079269 NR_003010 yes chr17 75082889 75087889 NR_003013 yes chr1 109640315 109645315 NR_003023 yes chr7 22893732 22898732 NR_003075 yes chr20 43989309 43994309 NR_003189 yes chr9 33621723 33626723 NR_003573 yes chr16 2388423 2393423 NR_003574 yes chr14 20792198 20797198 NR_003693 yes chr19 51105720 51110720 NR_004384 yes chr17 74555215 74560215 NR_004395 yes chr17 74554690 74559690 NR_004396 yes chr17 74552374 74557374 NR_004397 yes chr16 29872655 29877655 NR_015396 yes chr5 43039736 43044736 NR_015447 yes chr7 5011116 5016116 NR_015449 yes chr4 13546948 13551948 NR_015450 yes chr1 22349184 22354184 NR_023918 yes chr1 22349497 22354497 NR_023919 yes chr6 150323780 150328780 NR_024045 yes chr18 3591612 3596612 NR_024101 yes chr1 151808445 151813445 NR_024237 yes chr1 45767082 45772082 NR_024270 yes chr9 35906980 35911980 NR_024283 yes chr17 7816765 7821765 NR_024349 yes chr16 29872504 29877504 NR_024370 yes chr3 196666994 196671994 NR_024388 yes chr6 52526699 52531699 NR_024403 yes chr11 67082810 67087810 NR_024469 yes chr20 61295473 61300473 NR_024470 yes chr7 154717727 154722727 NR_024476 yes chr3 194028093 194033093 NR_024480 yes chr12 6860136 6865136 NR_026581 yes chr12 110208792 110213792 NR_026661 yes chr7 73146899 73151899 NR_026690 yes chr6 170187669 170192669 NR_026780 yes chr6 2985701 2990701 NR_026856 yes chr16 2890252 2895252 NR_026864 yes chr1 47897813 47902813 NR_026878 yes chr1 87595109 87600109 NR_026985 yes chr1 87592948 87597948 NR_026988 yes chr15 78284067 78289067 NR_026998 yes chr6 74017588 74022588 NR_027005 yes chr1 204336347 204341347 NR_027022 yes chr19 12200578 12205578 NR_027049 yes chr15 74419119 74424119 NR_027073 yes chr3 13689721 13694721 NR_027103 yes chr5 173004137 173009137 NR_027108 yes chr6 35702224 35707224 NR_027117 yes chr11 45790483 45795483 NR_027134 yes chr12 98907504 98912504 NR_027157 yes chr22 46399996 46404996 NR_027240 yes chr21 43526144 43531144 NR_027243 yes chr2 43451850 43456850 NR_027251 yes chr19 1267759 1272759 NR_027271 yes chr4 40056319 40061319 NR_027277 yes chr17 66192301 66197301 NR_027283 yes chr12 52602214 52607214 NR_027358 yes chr1 16969569 16974569 NR_027504 yes chr6 26419119 26424119 NR_027795 yes chr1 75196362 75201362 NR_027962 yes chr17 2316230 2321230 NR_028335 yes chr6 33548976 33553976 NR_028361 yes chr17 73630175 73635175 NR_028439 yes chr17 1417713 1422713 NR_028514 yes chr22 46507066 46512066 NR_029479 yes chr19 13944973 13949973 NR_029495 yes chr19 13944673 13949673 NR_029497 yes chr19 13944831 13949831 NR_029501 yes chr7 130559069 130564069 NR_029503 yes chr1 65521691 65526691 NR_029516 yes chr7 130559798 130564798 NR_029517 yes chr7 127845425 127850425 NR_029596 yes chr15 89152556 89157556 NR_029606 yes chr12 54383022 54388022 NR_029617 yes chr12 62994966 62999966 NR_029661 yes chr11 57406171 57411171 NR_029673 yes chr3 52325824 52330824 NR_029677 yes chr12 7070760 7075760 NR_029682 yes chr17 56406179 56411179 NR_029683 yes chr5 148805981 148810981 NR_029684 yes chr5 148807709 148812709 NR_029686 yes chr17 29884515 29889515 NR_029710 yes chr17 6918520 6923520 NR_029712 yes chr12 7070362 7075362 NR_029779 yes chr11 64656411 64661411 NR_029829 yes chr19 46139845 46144845 NR_029886 yes chr12 54728500 54733500 NR_029894 yes chr5 54463950 54468950 NR_029960 yes chr17 6918841 6923841 NR_030178 yes chr1 155162468 155167468 NR_030281 yes chr7 5533048 5538048 NR_030318 yes chr9 126162382 126167382 NR_030332 yes chr10 98586021 98591021 NR_030338 yes chr12 13066263 13071263 NR_030345 yes chr20 49199823 49204823 NR_030375 yes chr5 54464070 54469070 NR_030387 yes chr11 2015561 2020561 NR_030533 yes chr15 45722748 45727748 NR_030599 yes chr1 94309888 94314888 NR_030621 yes chr12 54425234 54430234 NR_030753 yes chr1 117211871 117216871 NR_031564 yes chr5 54465681 54470681 NR_031572 yes chr3 47888545 47893545 NR_031595 yes chr7 102043802 102048802 NR_031669 yes chr22 20071081 20076081 NR_031706 yes chr8 10680453 10685453 NR_031711 yes chr11 61580212 61585212 NR_031729 yes chr1 178509431 178514431 NR_033186 yes chr17 46667154 46672154 NR_033203 yes chr17 46670820 46675820 NR_033205 yes chr9 130878513 130883513 NR_033374 yes chr1 161065651 161070651 NR_033385 yes chr1 59248323 59253323 NR_034014 yes chr11 10560283 10565283 NR_034093 yes chr5 43064573 43069573 NR_034127 yes chr11 119249988 119254988 NR_034160 yes chr1 31189119 31194119 NR_034182 yes chr2 232575524 232580524 NR_036052_2 yes chr9 130545697 130550697 NR_036055 yes chr1 249118076 249123076 NR_036070 yes chr2 178118238 178123238 NR_036075 yes chr2 207645532 207650532 NR_036077 yes chr2 207645458 207650458 NR_036078 yes chr10 103358674 103363674 NR_036114 yes chr10 103358754 103363754 NR_036115 yes chr17 46799337 46804337 NR_036150 yes chr19 18390387 18395387 NR_036155 yes chr19 18494872 18499872 NR_036156 yes chr19 42635165 42640165 NR_036208 yes chr1 149819840 149824840 NR_036461 yes chr19 13944603 13949603 NR_036515 yes chr1 110879293 110884293 NR_036595 yes chr6 42692814 42697814 NR_037141 yes chr1 167187623 167192623 NR_037163 yes chr22 20070769 20075769 NR_037412 yes chr11 62325278 62330278 NR_037427 yes chr10 118924785 118929785 NR_037436 yes chr1 65521025 65526025 NR_037443 yes chr17 73399650 73404650 NR_037449 yes chr6 36587789 36592789 NR_037491 yes chr6 33663405 33668405 NR_037498 yes chr14 52115962 52120962 NR_037676 yes chr17 33413848 33418848 NR_037713 yes chr17 33446041 33451041 NR_037714 yes chr17 6913236 6918236 NR_037717 yes chr1 11897876 11902876 NR_037806 yes chr3 5019146 5024146 NR_037903 yes chr17 7462809 7467809 NR_037926 yes chr17 74551346 74556346 NR_038108 yes chr1 63780428 63785428 NR_038252 yes chr12 93962674 93967674 NR_038263 yes chr3 196727277 196732277 NR_038285 yes chr4 79564648 79569648 NR_038303 yes chr17 6920473 6925473 NR_038310 yes chr19 42634281 42639281 NR_038332 yes chr12 89410969 89415969 NR_038385 yes chr17 56403799 56408799 NR_038411 yes chr17 56404466 56409466 NR_038413 yes chr14 75758607 75763607 NR_038421 yes chr2 13144638 13149638 NR_038434 yes chr19 47985039 47990039 NR_038452 yes chr3 142643017 142648017 NR_038455 yes chr4 87853502 87858502 NR_038841 yes chr8 71518312 71523312 NR_038881 yes chr21 36115622 36120622 NR_038885 yes chr7 100199161 100204161 NR_038910 yes chr11 65335384 65340384 NR_038923 yes chr2 85763509 85768509 NR_038942 yes chr1 51523009 51528009 NR_039617 yes chr2 64750147 64755147 NR_039633 yes chr3 48235554 48240554 NR_039645 yes chr9 124879946 124884946 NR_039690 yes chr11 61273568 61278568 NR_039708 yes chr11 118778917 118783917 NR_039713 yes chr14 74222950 74227950 NR_039727 yes chr15 81287314 81292314 NR_039739 yes chr17 80623683 80628683 NR_039751 yes chr19 39897818 39902818 NR_039755 yes chr19 45154502 45159502 NR_039756 yes chr22 42316801 42321801 NR_039760 yes chr9 130545612 130550612 NR_039767 yes chr6 44219522 44224522 NR_039790 yes chr9 130629274 130634274 NR_039819 yes chr1 243506978 243511978 NR_039824 yes chr11 3874792 3879792 NR_039835 yes chr15 89152655 89157655 NR_039867 yes chr17 73778188 73783188 NR_039892 yes chr22 19948776 19953776 NR_039918 yes chr22 46506946 46511946 NR_039920 yes chr4 110352473 110357473 NR_039978 yes chr16 4301290 4306290 NR_039999 yes chr2 177499802 177504802 NR_040001 yes chr2 202020015 202025015 NR_040030 yes chr19 47162235 47167235 NR_040041 yes chr19 47161121 47166121 NR_040042 yes chr2 11269802 11274802 NR_040080 yes chr17 6913153 6918153 NR_040089 yes chr15 73072676 73077676 NR_040107 yes chr8 67338712 67343712 NR_040434 yes chr14 23282601 23287601 NR_040448 yes chr16 48387096 48392096 NR_040677 yes chr1 155021141 155026141 NR_040772 yes chr1 155033967 155038967 NR_040773 yes chr22 45018799 45023799 NR_044991 yes chr12 51439600 51444600 NR_045017 yes chr17 42296494 42301494 NR_045058 yes chr5 131743965 131748965 NR_045116 yes chr6 150182134 150187134 NR_045126 yes chr19 14245464 14250464 NR_045214 yes chr2 136575261 136580261 NR_045486 yes chr11 75477192 75482192 NR_046090 yes chr1 98512749 98517749 NR_046105 yes chr12 92532989 92537989 NR_046159 yes chr12 92534107 92539107 NR_046160 yes chr16 89230291 89235291 NR_046200 yes chr2 209117457 209122457 NR_046452 yes chr1 110750109 110755109 NR_046546 yes chr1 201796187 201801187 NR_046696 yes chr3 52321249 52326249 NR_046719 yes chr1 119909069 119914069 NR_046780 yes chr7 5717592 5722592 NR_046834 yes chr1 47642422 47647422 NR_047498 yes chr12 54376803 54381803 NR_047506 yes chr16 3079982 3084982 NR_047572 yes chr3 45548537 45553537 NR_048543 yes chr6 138186870 138191870 NR_049793 yes chr1 161194476 161199476 NR_049819 yes chr12 125397593 125402593 NR_049820 yes chr8 81151124 81156124 NR_049894 yes chr12 123754363 123759363 NR_073007 yes chr12 123753149 123758149 NR_073008 yes chr11 64214029 64219029 NR_073177 yes chr3 46596388 46601388 NR_073385 yes chr14 36537133 36542133 NR_073454 yes chr17 37821734 37826734 NR_073461 yes chr17 40825608 40830608 NR_073574 yes chr17 38181398 38186398 NR_102369 yes chr20 61434439 61439439 NR_102430 yes chr14 24503121 24508121 NR_102689 yes chr5 43064625 43069625 NR_102752 yes chr5 37838040 37843040 NR_103441 yes chr6 52439500 52444500 NR_103446 yes chr6 52439596 52444596 NR_103447 yes chr1 151988254 151993254 NR_103561 yes chr17 47648926 47653926 NR_103773 yes chr1 113613224 113618224 NR_103777 yes chr1 101699584 101704584 NR_104626 yes chr5 43065041 43070041 NR_104651 yes chr3 50262368 50267368 NR_106714 yes chr19 14181673 14186673 NR_106715 yes chr1 63790155 63795155 NR_106716 yes chr21 45027370 45032370 NR_106718 yes chr19 45937412 45942412 NR_106736 yes chr1 51523190 51528190 NR_106754 yes chr11 47198788 47203788 NR_106803 yes chr19 6734289 6739289 NR_106849 yes chr2 238417074 238422074 NR_106869 yes chr22 30400538 30405538 NR_106876 yes chr3 48584954 48589954 NR_106881 yes chr8 145538478 145543478 NR_106907 yes chr9 33465433 33470433 NR_106910 yes chr17 38347349 38352349 NR_106927 yes chr3 50308167 50313167 NR_106932 yes chr15 75130548 75135548 NR_106942 yes chr17 38180162 38185162 NR_106944 yes chr6 35435785 35440785 NR_106961 yes chr11 64106876 64111876 NR_106977 yes chr14 72981028 72986028 NR_106994 yes chr17 75082999 75087999 NR_106997 yes chr11 1898775 1903775 NR_107001 yes chr12 123846890 123851890 NR_107039 yes chr10 17269484 17274484 NR_108061 yes chr8 128410144 128415144 NR_109834 yes chr20 39764140 39769140 NR_109889 yes chr20 43076697 43081697 NR_109893 yes chr5 5137667 5142667 NR_109915 yes chr14 23396318 23401318 NR_110002 yes chr20 49545021 49550021 NR_110007 yes chr12 124066374 124071374 NR_110049 yes chr2 179275886 179280886 NR_110204 yes chr2 65088265 65093265 NR_110224 yes chr14 24027806 24032806 NR_110555 yes chr2 177499982 177504982 NR_110599 yes chr19 56575550 56580550 NR_110741 yes chr17 63094430 63099430 NR_110801 yes chr17 76354029 76359029 NR_110845 yes chr17 76354583 76359583 NR_110846 yes chr16 4375841 4380841 NR_110901 yes chr16 47175479 47180479 NR_110903 yes chr1 245131671 245136671 NR_111907 yes chr8 128743713 128748713 NR_117101 yes chr16 87810254 87815254 NR_120309 yes chr15 45568920 45573920 NR_120335 yes chr15 68128717 68133717 NR_120345 yes chr12 52476030 52481030 NR_120438 yes chr12 10900333 10905333 NR_120463 yes chr11 78138382 78143382 NR_120564 yes chr11 124629827 124634827 NR_120579 yes chr11 66245220 66250220 NR_120586 yes chr8 144361370 144366370 NR_120682 yes chr9 77565381 77570381 NR_121183 yes chr4 40306702 40311702 NR_121641 yes chr10 118926066 118931066 NR_121650 yes chr1 46912876 46917876 NR_121680 yes chr12 62994714 62999714 NR_121682 yes chr7 91761377 91766377 NR_122109 yes chr16 3106871 3111871 NR_123723 yes chr3 178863261 178868261 NR_125401 yes chr8 80677877 80682877 NR_125410 yes chr8 10695036 10700036 NR_125432 yes chr20 48806106 48811106 NR_125739 yes chr11 125032005 125037005 NR_125759 yes chr1 85084214 85089214 NR_125761 yes chr6 1603266 1608266 NR_125804 yes chr6 44039889 44044889 NR_125864 yes chr1 95697038 95702038 NR_125948 yes chr1 26791528 26796528 NR_125952 yes chr1 112295631 112300631 NR_125963 yes chr1 47900489 47905489 NR_126355 yes chr17 73994740 73999740 NR_130467_2 yes chr10 102898523 102903523 NR_130724 yes chr19 46142252 46147252 NR_130728 yes chr5 139534404 139539404 NR_130738 yes chr8 130689985 130694985 NR_130917 yes chr15 85194977 85199977 NR_130944 yes chr14 50569261 50574261 NR_131171 yes chr11 2020196 2025196 NR_131224 yes chr20 30306810 30311810 NR_131907 yes chr7 22764739 22769739 NR_131935 yes chr9 132478485 132483485 NR_132102 yes chr19 48269597 48274597 NR_132382 yes chr1 9239763 9244763 NR_132738 yes chr1 9239897 9244897 NR_132742 yes chr1 39978038 39983038 NR_132962 yes chr11 1968061 1973061 NR_ 132974 yes chr6 74225661 74230661 NR_132980_2 yes chr16 14393645 14398645 NR_ 132983 yes chr16 14393862 14398862 NR_ 132984 yes chr11 64658421 64663421 NR_133638 yes chr21 44558702 44563702 NR_133677 yes chr20 30307633 30312633 NR_134257 yes chr5 10519696 10524696 NR_134289 yes chr8 145594148 145599148 NR_134307 yes chr9 35113469 35118469 NR_134455 yes chr16 30438621 30443621 NR_134471 yes chr6 150253149 150258149 NR_134598 yes chr6 26567052 26572052 NR_134611 yes chr6 33599022 33604022 NR_134628 yes chr19 51286967 51291967 NR_134883 yes chr19 18313040 18318040 NR_134910 yes chr3 195584775 195589775 NR_134939 yes chr12 47698682 47703682 NR_135024 yes chr12 7069908 7074908 NR_135032 yes chr12 92537361 92542361 NR_135036 yes chr1 91314695 91319695 NR_135038 yes chr12 122498696 122503696 NR_135044 yes chr10 102823556 102828556 NR_135068 yes chr12 6996022 7001022 NR_135083 yes chr14 91161177 91166177 NR_135190 yes chr14 90981053 90986053 NR_135274 yes chr3 72710900 72715900 NR_135531 yes chr2 101765622 101770622 NR_135594 yes chr1 212000479 212005479 NR_135818 yes chr1 212000688 212005688 NR_135819 yes chr1 212001803 212006803 NR_ 135820 yes chr15 41196507 41201507 NR_135836 yes chr1 87167676 87172676 NR_135837 yes chr10 98589715 98594715 NR_135921 yes chr2 64748789 64753789 NR_136167 yes chr6 16758869 16763869 NR_136240 yes chr17 7484897 7489897 NR_136401 yes chr11 2321779 2326779 NR_138249 yes chr6 36643836 36648836 NR_144384 yes chr17 40704320 40709320 NR_144402 yes chr17 48276500 48281500 NM_000088 no chr16 67462536 67467536 NM_000196 no chr17 45328708 45333708 NM_000212 no chr4 100482735 100487735 NM_000253 no chr11 76836810 76841810 NM_000260 no chr3 38688663 38693663 NM_000335 no chr16 56896619 56901619 NM_000339 no chr20 23027801 23032801 NM_000361 no chr12 48296314 48301314 NM_000376 no chr1 21833351 21838351 NM_000478 no chr7 45958371 45963371 NM_000598 no chr8 18065112 18070112 NM_000662 no chr5 159341240 159346240 NM_000679 no chr11 15092646 15097646 NM_000728 no chr11 62686512 62691512 NM_000738 no chr11 46405658 46410658 NM_000741 no chr5 174868663 174873663 NM_000794 no chr10 74854232 74859232 NM_000917 no chr21 45717417 45722417 NM_001002021 no chr2 96801675 96806675 NM_001002036 no chr7 150943249 150948249 NM_001003801 no chr2 71015275 71020275 NM_001004311 no chr1 242159885 242164885 NM_001004343 no chr11 4658445 4663445 NM_001004751 no chr17 57406553 57411553 NM_001005404 no chr6 130337014 130342014 NM_001007102 no chr20 23584110 23589110 NM_001008693 no chr8 30239517 30244517 NM_001008710 no chr10 106111022 106116022 NM_001008723 no chr19 9692709 9697709 NM_001008727 no chr2 158181725 158186725 NM_001009959 no chr10 21783713 21788713 NM_001010911 no chr20 45310624 45315624 NM_001011554 no chr3 118751176 118756176 NM_001015887 no chr4 38782111 38787111 NM_001017388 no chr22 41937993 41942993 NM_001018050 no chr8 56984640 56989640 NM_001023 no chr1 6301752 6306752 NM_001024598 no chr19 43030161 43035161 NM_001024912 no chr12 50014697 50019697 NM_001031698 no chr4 159091702 159096702 NM_001031700 no chr3 9288869 9293869 NM_001033117 no chr11 14991332 14996332 NM_001033952 no chr2 10260363 10265363 NM_001034 no chr12 51816094 51821094 NM_001039960 no chr1 212779470 212784470 NM_001040619 no chr19 41100641 41105641 NM_001042544 no chr19 41104777 41109777 NM_001042545 no chr19 45346893 45351893 NM_001042724 no chr2 219132615 219137615 NM_001077399 no chr19 51520931 51525931 NM_001077500 no chr8 133069127 133074127 NM_001080399 no chr14 75328037 75333037 NM_001080408 no chr7 1541518 1546518 NM_001080453 no chr2 85105869 85110869 NM_001080824 no chr16 3505485 3510485 NM_001083600 no chr2 219132432 219137432 NM_001087 no chr17 74461130 74466130 NM_001088 no chr6 35106687 35111687 NM_001093728 no chr14 21154432 21159432 NM_001097577 no chr11 60521840 60526840 NM_001098835 no chr2 70778647 70783647 NM_001099691 no chr22 24638610 24643610 NM_001099781 no chr16 30933396 30938396 NM_001099784 no chr19 18696995 18701995 NM_001100418 no chr12 109824024 109829024 NM_001101421 no chr16 30668340 30673340 NM_001105079 no chr7 75828711 75833711 NM_001110199 no chr15 89784694 89789694 NM_001113378 no chr11 76836816 76841816 NM_001127180 no chr22 18558260 18563260 NM_001127649 no chr11 47427546 47432546 NM_001128225 no chr8 22222262 22227262 NM_001128431 no chr3 51702691 51707691 NM_001129884 no chr10 99329698 99334698 NM_001129981 no chr17 36828687 36833687 NM_001130677 no chr4 122870409 122875409 NM_001130698 no chr7 129012984 129017984 NM_001130722 no chr7 129005464 129010464 NM_001130723 no chr3 71775769 71780769 NM_001134650 no chr4 100482714 100487714 NM_001134665 no chr8 22222550 22227550 NM_001135153 no chr17 19646425 19651425 NM_001135167 no chr17 19646272 19651272 NM_001135168 no chr2 88749553 88754553 NM_001135649 no chr8 20038217 20043217 NM_001135691 no chr1 15476528 15481528 NM_001136216 no chr1 15477729 15482729 NM_001136218 no chr11 118013684 118018684 NM_001142349 no chr11 27720100 27725100 NM_001143808 no chr11 27719947 27724947 NM_001143812 no chr11 27719535 27724535 NM_001143813 no chr11 72351004 72356004 NM_001143839 no chr5 142075135 142080135 NM_001144934 no chr12 56099186 56104186 NM_001144996 no chr11 43961606 43966606 NM_001145033 no chr9 133811955 133816955 NM_001145106 no chr9 114359635 114364635 NM_001146108 no chr21 40757192 40762192 NM_001146218 no chr16 8619726 8624726 NM_001146336 no chr17 40994100 40999100 NM_001158 no chr5 173041166 173046166 NM_001159651 no chr3 38688664 38693664 NM_001160160 no chr15 44953376 44958376 NM_001160227 no chr16 18993109 18998109 NM_001160364 no chr19 51842878 51847878 NM_001163922 no chr2 10260195 10265195 NM_001165931 no chr17 48346267 48351267 NM_001168215 no chr3 119960642 119965642 NM_001168271 no chr6 12288029 12293029 NM_001168319 no chr3 137829822 137834822 NM_001170538 no chr3 51531518 51536518 NM_001171904 no chr10 73569140 73574140 NM_001171935 no chr7 116162563 116167563 NM_001172895 no chr7 116163847 116168847 NM_001172896 no chr7 116163912 116168912 NM_001172897 no chr9 117265236 117270236 NM_001173425 no chr20 46128101 46133101 NM_001174087 no chr17 26938711 26943711 NM_001174103 no chr12 51982520 51987520 NM_001177984 no chr12 859589 864589 NM_001184985 no chr2 234157717 234162717 NM_001190266 no chr10 106032387 106037387 NM_001191014 no chr12 113571544 113576544 NM_001193520 no chr12 113571059 113576059 NM_001193521 no chr16 58032777 58037777 NM_001195302 no chr11 74949450 74954450 NM_001195528 no chr17 74862298 74867298 NM_001199172 no chr9 117441869 117446869 NM_001199233 no chr22 18558186 18563186 NM_001199319 no chr19 45752016 45757016 NM_001199867 no chr16 71915593 71920593 NM_001201552 no chr16 71914944 71919944 NM_001201553 no chr4 155469085 155474085 NM_001201564 no chr5 172568945 172573945 NM_001205 no chr2 219260561 219265561 NM_001206878 no chr9 34587638 34592638 NM_001207011 no chr22 31476346 31481346 NM_001207017 no chr22 31478482 31483482 NM_001207018 no chr18 30047947 30052947 NM_001242409 no chr3 51702790 51707790 NM_001243725 no chr11 120104849 120109849 NM_001244682 no chr5 39422835 39427835 NM_001244871 no chr15 51198369 51203369 NM_001252127 no chr2 27068657 27073657 NM_001253723 no chr2 27068792 27073792 NM_001253724 no chr2 31359092 31364092 NM_001253826 no chr18 20712028 20717028 NM_001256438 no chr2 128403328 128408328 NM_001256542 no chr5 73934749 73939749 NM_001256574 no chr12 121645164 121650164 NM_001256796 no chr1 2123714 2128714 NM_001256945 no chr12 51816401 51821401 NM_001258403 no chr7 23747445 23752445 NM_001260504 no chr7 23747286 23752286 NM_001260505 no chr17 79978285 79983285 NM_001271006 no chr5 1006577 1011577 NM_001271082 no chr10 32633646 32638646 NM_001272004 no chr10 32633476 32638476 NM_001272019 no chr9 139375711 139380711 NM_001276418 no chr19 42298022 42303022 NM_001277163 no chr2 72372491 72377491 NM_001277742 no chr19 17389954 17394954 NM_001278443 no chr22 24817065 24822065 NM_001278500 no chr6 159460885 159465885 NM_001278733 no chr17 47782813 47787813 NM_001278784 no chr17 15163406 15168406 NM_001281455 no chr16 30931876 30936876 NM_001282351 no chr9 137977014 137982014 NM_001282611 no chr9 137976606 137981606 NM_001282612 no chr2 10970111 10975111 NM_001282704 no chr2 10975603 10980603 NM_001282705 no chr22 31157740 31162740 NM_001282740 no chr22 41938110 41943110 NM_001282884 no chr2 232648593 232653593 NM_001282950 no chr9 135283083 135288083 NM_001282957 no chr2 97168622 97173622 NM_001285485 no chr19 33790970 33795970 NM_001285829 no chr21 30462075 30467075 NM_001286619 no chr9 132379992 132384992 NM_001286797 no chr9 132385932 132390932 NM_001286798 no chr9 132386223 132391223 NM_001286799 no chr16 8617507 8622507 NM_001290095 no chr19 42210004 42215004 NM_001291484 no chr22 22897674 22902674 NM_001291715 no chr22 22899268 22904268 NM_001291717 no chr5 73933348 73938348 NM_001292004 no chr11 69059105 69064105 NM_001293291 no chr15 81069184 81074184 NM_001293298 no chr3 46247332 46252332 NM_001295 no chr4 8268992 8273992 NM_001297559 no chr1 244813850 244818850 NM_001297746 no chr11 58936403 58941403 NM_001300727 no chr16 18992756 18997756 NM_001300732 no chr1 234347457 234352457 NM_001300845 no chr11 66821789 66826789 NM_001300886 no chr11 107433961 107438961 NM_001301010 no chr5 148518510 148523510 NM_001301015 no chr19 41768455 41773455 NM_001301016 no chr15 45026060 45031060 NM_001301144 no chr15 78110742 78115742 NM_001301186 no chr15 78110174 78115174 NM_001301187 no chr15 78109944 78114944 NM_001301189 no chr15 78109366 78114366 NM_001301191 no chr15 78076856 78081856 NM_001301195 no chr12 113570904 113575904 NM_001301202 no chr17 41558785 41563785 NM_001302623 no chr9 100171802 100176802 NM_001302884 no chr18 3245028 3250028 NM_001303047 no chr18 3244980 3249980 NM_001303048 no chr3 73480647 73485647 NM_001303142 no chr22 39265839 39270839 NM_001303494 no chr2 158181321 158186321 NM_001304344 no chr2 158179960 158184960 NM_001304346 no chr20 1162819 1167819 NM_001304748 no chr20 1163577 1168577 NM_001304749 no chr11 122730519 122735519 NM_001304782 no chr4 5050744 5055744 NM_001306082 no chr5 118321415 118326415 NM_001308081 no chr2 70778270 70783270 NM_001308158 no chr3 155521576 155526576 NM_001308229 no chr10 129702825 129707825 NM_001316676 no chr19 59028426 59033426 NM_001316978 no chr19 59028876 59033876 NM_001316979 no chr8 23101605 23106605 NM_001317899 no chr20 50416562 50421562 NM_001318031 no chr22 22895847 22900847 NM_001318126 no chr10 28589495 28594495 NM_001318170 no chr16 71915575 71920575 NM_001318238 no chr16 71911879 71916879 NM_001318239 no chr4 128649033 128654033 NM_001318467 no chr1 15477707 15482707 NM_001319665 no chrX 15516400 15521400 NM_001320866 no chr19 41767764 41772764 NM_001321208 no chr17 35730428 35735428 NM_001321399 no chr9 4982586 4987586 NM_001322194 no chr9 4982730 4987730 NM_001322196 no chr17 41558733 41563733 NM_001322216 no chr5 178155203 178160203 NM_001324339 no chr14 23338426 23343426 NM_001329226 no chr1 233083777 233088777 NM_001329452 no chr5 90676690 90681690 NM_001329672 no chr16 83999737 84004737 NM_001329748 no chr2 70526677 70531677 NM_001329752 no chr2 70526715 70531715 NM_001329753 no chr2 70526720 70531720 NM_001329755 no chr14 76445865 76450865 NM_001329938 no chr14 76446854 76451854 NM_001329939 no chr11 47427551 47432551 NM_001330245 no chr16 19176656 19181656 NM_001330509 no chr17 79978294 79983294 NM_001330536 no chr16 58031003 58036003 NM_001330568 no chr12 64236033 64241033 NM_001346201 no chr6 130338289 130343289 NM_001346550 no chr6 130337705 130342705 NM_001346551 no chr15 51571959 51576959 NM_001347251 no chr4 55092756 55097756 NM_001347827 no chr4 55096749 55101749 NM_001347829 no chr4 55097627 55102627 NM_001347830 no chr5 111752513 111757513 NM_001347887 no chr8 28556490 28561490 NM_001440 no chr8 82190218 82195218 NM_001444 no chr3 49708935 49713935 NM_001640 no chr4 75308315 75313315 NM_001657 no chr4 75478091 75483091 NM_001657_2 no chr5 78405104 78410104 NM_001713 no chr16 71390116 71395116 NM_001740 no chr7 116162339 116167339 NM_001753 no chr9 34587235 34592235 NM_001842 no chr16 21311957 21316957 NM_001888 no chr2 10440530 10445530 NM_002149 no chr2 219922738 219927738 NM_002181 no chr2 11049563 11054563 NM_002236 no chr11 8282925 8287925 NM_002315 no chr11 46937673 46942673 NM_002334 no chr11 61973640 61978640 NM_002407 no chr19 42256928 42261928 NM_002483 no chr17 3817460 3822460 NM_002558 no chr1 2341510 2346510 NM_002617 no chr20 52822002 52827002 NM_002623 no chr19 51520782 51525782 NM_002776 no chr11 119596935 119601935 NM_002855 no chr17 32687752 32692752 NM_002981 no chr8 134581683 134586683 NM_003033 no chr6 50783939 50788939 NM_003221 no chr15 39870780 39875780 NM_003246 no chr4 38803912 38808912 NM_003263 no chr17 34133959 34138959 NM_003487 no chr8 23019043 23024043 NM_003840 no chr2 86113657 86118657 NM_003896 no chr6 158400388 158405388 NM_003898 no chr22 23520052 23525052 NM_004327 no chr6 75913123 75918123 NM_004370 no chrX 68046340 68051340 NM_004429 no chr3 184277087 184282087 NM_004443 no chr9 4982745 4987745 NM_004972 no chr20 48096681 48101681 NM_004975 no chr17 48070088 48075088 NM_005220 no chr10 120964697 120969697 NM_005308 no chr10 93390358 93395358 NM_005398 no chr17 54668560 54673560 NM_005450 no chr17 40910712 40915712 NM_005854 no chr3 40348673 40353673 NM_005875 no chr9 36166889 36171889 NM_005893 no chr6 137111156 137116156 NM_005923 no chr10 111983262 111988262 NM_005962 no chr2 232392682 232397682 NM_006056 no chr4 38855938 38860938 NM_006068 no chr11 76811386 76816386 NM_006189 no chr19 18630437 18635437 NM_006532 no chr16 24264374 24269374 NM_006539 no chr9 92217427 92222427 NM_006705 no chr2 118569755 118574755 NM_006773 no chr22 31474782 31479782 NM_006932 no chr19 41767579 41772579 NM_007040 no chr5 95295275 95300275 NM_012081 no chr6 3847100 3852100 NM_012135 no chr11 33561377 33566377 NM_012194 no chr9 114359211 114364211 NM_012212 no chr12 50014903 50019903 NM_012272 no chr10 71208726 71213726 NM_012339 no chr20 4700000 4705000 NM_012409 no chr16 69342787 69347787 NM_013245 no chr6 16126817 16131817 NM_013262 no chr19 40929432 40934432 NM_013376 no chr8 53319939 53324939 NM_014682 no chr9 139375007 139380007 NM_014866 no chr4 99914288 99919288 NM_015143 no chr14 103056496 103061496 NM_015156 no chr11 58937312 58942312 NM_015177 no chr17 5971434 5976434 NM_015253 no chr5 173470107 173475107 NM_015980 no chr5 149666903 149671903 NM_015981 no chr16 19177035 19182035 NM_016524 no chr5 33933991 33938991 NM_016568 no chr4 159091218 159096218 NM_016613 no chr21 26977301 26982301 NM_017446 no chr14 76042440 76047440 NM_017791 no chr1 245315787 245320787 NM_018012 no chr20 1162617 1167617 NM_018354 no chr12 68617071 68622071 NM_018402 no chr19 4789228 4794228 NM_018708 no chr3 56833495 56838495 NM_019555 no chr20 54821288 54826288 NM_019888 no chr2 27068469 27073469 NM_020134 no chr3 52476601 52481601 NM_020163 no chr7 105514531 105519531 NM_020725 no chr6 40552703 40557703 NM_020737 no chr6 44278563 44283563 NM_020745 no chr16 58031262 58036262 NM_020807 no chr20 22562601 22567601 NM_021784 no chr20 43371988 43376988 NM_022358 no chr12 118811858 118816858 NM_022491 no chr6 119397312 119402312 NM_024581 no chr15 101456920 101461920 NM_024652 no chr9 27527350 27532350 NM_024761 no chr3 133646156 133651156 NM_025041 no chr1 208415165 208420165 NM_025179 no chr19 41033895 41038895 NM_025213 no chr20 31592884 31597884 NM_025227 no chr17 41833656 41838656 NM_025237 no chr19 10809612 10814612 NM_031209 no chr2 85358083 85363083 NM_031283 no chr1 6482348 6487348 NM_031475 no chr12 57846574 57851574 NM_031479 no chr11 94797541 94802541 NM_032102 no chr19 11667551 11672551 NM_032377 no chr1 20957448 20962448 NM_032409 no chr17 72207196 72212196 NM_032646 no chr9 133811739 133816739 NM_032843 no chr11 12305947 12310947 NM_032867 no chr9 116915325 116920325 NM_032888 no chr5 141991226 141996226 NM_033137 no chr22 20305128 20310128 NM_033257 no chr11 69453373 69458373 NM_053056 no chr6 159463684 159468684 NM_054114 no chr19 10044728 10049728 NM_058164 no chr12 55245799 55250799 NM_058173 no chr22 46370508 46375508 NM_058238 no chr22 31501073 31506073 NM_080430 no chr20 31589739 31594739 NM_080675 no chr20 44255885 44260885 NM_080753 no chr10 111964863 111969863 NM_130439 no chr19 3583069 3588069 NM_133261 no chr4 1899853 1904853 NM_133334 no chr2 10441326 10446326 NM_134421 no chr20 23417822 23422822 NM_138283 no chr14 105442194 105447194 NM_138420 no chr6 151644166 151649166 NM_144497 no chr12 8972568 8977568 NM_144670 no chr19 41765881 41770881 NM_144732 no chr17 79978678 79983678 NM_144999 no chr10 71387503 71392503 NM_145306 no chr19 51520454 51525454 NM_145888 no chr20 44257407 44262407 NM_147198 no chr22 37953971 37958971 NM_152243 no chr8 23098650 23103650 NM_152272 no chr4 100482325 100487325 NM_152292 no chr11 100555907 100560907 NM_152432 no chr21 40813628 40818628 NM_152505 no chr9 139375447 139380447 NM_152571 no chr17 48349288 48354288 NM_153229 no chr17 15161661 15166661 NM_153322 no chr20 22563601 22568601 NM_153675 no chr11 27720680 27725680 NM_170733 no chr11 27718714 27723714 NM_170734 no chr10 103597111 103602111 NM_173194 no chr9 138232595 138237595 NM_173520 no chr3 137831951 137836951 NM_173543 no chr19 51758464 51763464 NM_173635 no chr5 118321800 118326800 NM_173666 no chr9 126115946 126120946 NM_173689 no chr7 150145218 150150218 NM_175571 no chr20 31365158 31370158 NM_175850 no chr17 79916557 79921557 NM_178493 no chr19 52640691 52645691 NM_178523 no chr6 47747275 47752275 NM_181744 no chr20 31237283 31242283 NM_182584 no chr14 21154436 21159436 NM_194431 no chr19 660733 665733 NM_194460 no chr5 153567795 153572795 NM_198321 no chr16 85143614 85148614 NM_198491 no chr22 36016901 36021901 NM_203377 no chr2 33169869 33174869 NM_206943 no chr22 39707416 39712416 NR_000028 no chr8 56983960 56988960 NR_002437 no chr14 101121105 101126105 NR_024096 no chr17 48247337 48252337 NR_024192 no chr15 37176234 37181234 NR_024264 no chr12 121407595 121412595 NR_024345 no chr16 30932090 30937090 NR_024348 no chr11 122071317 122076317 NR_024430 no chr17 40910775 40915775 NR_024461 no chr16 21309670 21314670 NR_026675 no chr9 100156473 100161473 NR_026847 no chr19 33791263 33796263 NR_026887 no chr17 72206960 72211960 NR_026914 no chr22 31315795 31320795 NR_026920 no chr1 24524230 24529230 NR_027087 no chr1 15476460 15481460 NR_027136 no chr22 22899250 22904250 NR_027426 no chr5 111752780 111757780 NR_027706 no chr2 219863930 219868930 NR_029867 no chr10 21783070 21788070 NR_031736 no chr15 93336214 93341214 NR_033769 no chr1 20752787 20757787 NR_033887 no chr3 40348689 40353689 NR_033965 no chr12 127356736 127361736 NR_033970 no chr2 219920972 219925972 NR_036081 no chr1 23187219 23192219 NR_036214 no chr15 79041879 79046879 NR_036495 no chr12 111371906 111376906 NR_036513 no chr7 99575885 99580885 NR_036679 no chr4 99916038 99921038 NR_037455 no chr17 79280548 79285548 NR_038080 no chr2 86039753 86044753 NR_038888 no chr1 19207269 19212269 NR_039844 no chr1 235673625 235678625 NR_039973_2 no chr2 219864437 219869437 NR_046086 no chr16 66439927 66444927 NR_046242 no chr12 9783515 9788515 NR_046448 no chr21 30742321 30747321 NR_046564 no chr3 156162225 156167225 NR_046618 no chr5 142245975 142250975 NR_046680 no chr13 31374843 31379843 NR_047012 no chr11 4205882 4210882 NR_047550 no chr2 182816468 182821468 NR_048567 no chr22 23225947 23230947 NR_049835 no chr3 185483192 185488192 NR_049838_2 no chr3 51703143 51708143 NR_103462 no chr4 41882128 41887128 NR_104143 no chr10 32633792 32638792 NR_104163 no chr14 77250567 77255567 NR_104183 no chr12 54148318 54153318 NR_104332 no chr2 97171346 97176346 NR_104346 no chr1 20957672 20962672 NR_106732 no chr17 47363316 47368316 NR_106745 no chr2 218762851 218767851 NR_106867 no chr12 121879655 121884655 NR_106957 no chr10 106110833 106115833 NR_108036 no chr20 21548162 21553162 NR_109880 no chr5 79376988 79381988 NR_109930 no chr16 66507805 66512805 NR_109960 no chr12 103939072 103944072 NR_110103 no chr12 106095481 106100481 NR_110108 no chr12 106098052 106103052 NR_110110 no chr2 71173241 71178241 NR_110273 no chr11 94881203 94886203 NR_110303 no chr7 55320828 55325828 NR_110426 no chr14 76043431 76048431 NR_110552 no chr2 86113903 86118903 NR_110569 no chr16 5663751 5668751 NR_110902 no chr15 77949893 77954893 NR_120361 no chr11 119597793 119602793 NR_120587 no chr4 40316002 40321002 NR_121640 no chr3 138660362 138665362 NR_121649 no chr11 45390448 45395448 NR_122071 no chr12 8831773 8836773 NR_123740 no chr19 46578387 46583387 NR_125344 no chr10 90689941 90694941 NR_125373 no chr11 130729505 130734505 NR_125383 no chr8 134896239 134901239 NR_125424 no chr12 4382850 4387850 NR_125790 no chr8 28556481 28561481 NR_126027 no chr11 72279200 72284200 NR_126364 no chr13 76448522 76453522 NR_126373 no chr17 67228653 67233653 NR_130736 no chr5 95295205 95300205 NR_130776 no chr11 43588356 43593356 NR_131246 no chr2 192552407 192557407 NR_131917 no chr20 43372368 43377368 NR_132377 no chr15 89902310 89907310 NR_133001 no chr11 100556186 100561186 NR_133571 no chr11 47426327 47431327 NR_134854 no chr19 11311804 11316804 NR_134909 no chr11 94471021 94476021 NR_135093 no chr15 101456988 101461988 NR_135827 no chr20 55150119 55155119 NR_136537 no chr1 233084088 233089088 NR_138027 no

Example 2—MYC Enhancer-Docking Site

Previous studies have established that tumor cells acquire tumor-specific super-enhancers at various sites throughout the MYC locus (FIG. 1B, 7A) (Bradner et al., 2017; Chapuy et al., 2013; Gabay et al., 2014; Gröschel et al., 2014; Herranz et al., 2014; Hnisz et al., 2013; Lin et al., 2016; Lovén et al., 2013; Parker et al., 2013; Zhang et al., 2015), but the mechanisms by which these diverse enhancer structures control MYC are not clear. To gain insights into the potential role of DNA loop structures in gene control at the MYC locus, we generated cohesin HiChIP data for HCT-116 cells and collected published DNA interaction data for three other cancer cell types for comparison (FIG. 1C)(Hnisz et al., 2016a; Pope et al., 2014). Among the DNA loop structures observed in these datasets, a large DNA loop was evident, spanning 2.8 Mb, that connects CTCF sites encompassing the MYC gene. The DNA anchor sites of this 2.8 Mb DNA loop occur at the boundaries of a TAD found in all cells (FIG. 7B). The MYC TAD encompasses a region previously described as a “gene desert” because this large span of DNA contains no other annotated protein-coding genes (Montavon and Duboule, 2012; Ovcharenko et al., 2005).

While all cells examined appear to share the TAD-spanning 2.8 Mb loop encompassing MYC, the loop structures within the neighborhood were found to be markedly different among the tumor types. The internal loops were dominated by interactions between a MYC promoter-proximal CTCF site and diverse cell specific super-enhancers (FIG. 1C). The major differences between these internal structures in the different tumor cells involved the different positions of the tumor-specific super-enhancer elements. Examination of Hi-C data for a broader spectrum of tumor cell types suggests that tumor cells generally have DNA contacts between the MYC promoter-proximal site and other sites within the 2.8 Mb MYC TAD (FIG. 7B).

Further examination of the MYC promoter-proximal region revealed three constitutive CTCF binding sites (FIG. 1D). All three sites were found to be occupied by CTCF in a wide variety of normal cells and tumor cells, and this binding pattern is shared across species (FIG. 1C). Previous studies have examined the role of CTCF binding at all three sites (Filippova et al., 1996; Gombert et al., 2003; Klenova et al., 1993, 2001; Rubio et al., 2008). The two sites located within the MYC gene have been shown to play roles in MYC transcript start site selection and in promoter-proximal pausing of RNA polymerase II (Filippova et al., 1996; Klenova et al., 2001). The CTCF binding site located 2 kb upstream of the major transcript start site, has been reported to be an insulator element (Gombert et al., 2003). The DNA interaction data described here, however, suggests that this upstream site dominates connections with distal enhancer elements, as the majority of reads in the DNA interaction data are associated with this site in all tumor cells examined (FIG. 1D, FIG. 7D). The −2 kb CTCF binding site contains a number of putative CTCF binding motifs; one of these most closely matches the canonical CTCF motif in the JASPAR database (Sandelin, 2004) and occurs within a highly conserved sequence (FIG. 1E, FIG. 7E). These features, the presence of CTCF sites in tumor super-enhancers and the ability of two CTCF-bound sites to be brought together through CTCF homodimerization (Saldana-Meyer et al., 2014; Yusufzai et al., 2004), led us to further study the possibility that the −2 kb site has an enhancer-docking function critical to MYC expression (FIG. 2A).

To determine whether the putative enhancer-docking site plays a functional role in MYC expression through DNA loop formation, we used the CRISPR/Cas9 system to delete a 210 bp segment centered on this site in the Chronic Myeloid Leukemia (CML) cell line K562 (FIG. 2B). Cells were virally transduced with constructs carrying Cas9 and doxycycline dependent gRNA expression cassettes, selected for the presence of the constructs, induced for gRNA expression, and harvested. Genotyping of the AK562 cells indicated that the putative enhancer-docking site was altered in approximately half of the alleles in the cell population (FIG. 8A). CTCF occupancy, measured in the total AK562 cell population by ChIP-seq, was reduced by approximately 2-fold at the site, while other sites, such as the comparable CTCF-bound region of the MYCL promoter, were unaffected (FIG. 2B). There was an approximately 2-fold reduction in MYC mRNA levels in the AK562 cells, indicating that the putative enhancer loop-anchor is necessary for the high levels of MYC expression normally produced by these cancer cells (FIG. 2C).

If the putative MYC enhancer-docking site contributes to looping interactions with distal enhancers, then the loss of this site in AK562 cells should cause a decrease in DNA interactions between the MYC promoter and the distal super-enhancers located nearly 2 Mb away in the downstream portion of the insulated neighborhood. We used chromosome conformation capture combined with high-throughput sequencing (4C-seq) to compare the interactions in K562 and AK562 cells (FIG. 2D, FIG. 8B). In wild-type cells, the 4C-seq data indicated that the putative enhancer-docking site interacts predominantly with the two distal super-enhancers (FIG. 2D) and that interactions between the enhancer-docking site and other DNA sites terminates at the TAD boundaries (FIG. 8B), indicating that the 2.8 Mb CTCF-CTCF loop has the properties expected of an insulted neighborhood (Dowen et al., 2014; Hnisz et al., 2016b). The results also showed that there was decreased interaction between the putative docking site and the distal super-enhancers in AK562 cells (FIG. 2D, FIG. 8B). This indicates that the CTCF site in the MYC promoter is important for optimal interaction with these distal enhancers and supports the idea that this CTCF site functions as an enhancer-docking site.

If the MYC enhancer-docking site functions similarly across a variety of tumor cells, then deletion of the site in various tumor cells should consistently cause reduced MYC expression. Indeed, when the CRISPR/Cas9 system was used to delete the 210 bp segment in colorectal cancer cells (HCT-116), acute T-cell leukemia cells (Jurkat) and breast cancer cells (MCF7), we consistently observed a reduction in MYC transcripts (FIG. 8C, D). The reduced expression of MYC in all these tumor types was accompanied by reduced cell proliferation in culture (FIG. 8E). These results suggest that MYC expression is similarly dependent on the MYC enhancer-docking site in multiple tumor cell types.

Importance of CTCF Motif Sequence in Enhancer-Docking Site

The enhancer-docking site contains multiple putative CTCF motifs, of which one stood out in terms of conservation and JASPAR score (FIG. 1E, 7E). To ascertain whether this CTCF binding site contributes to optimal MYC expression, small perturbations of the site were generated in both alleles of the tumor cell lines K562, HCT-116, Jurkat and MCF7 using CRISPR/Cas9 (FIG. 3A, B). Previous experiments were conducted on cell populations where the cells could survive reduced MYC levels if they suffered alteration of only one allele. In contrast, these CTCF binding site deletion experiments were conducted in cells with an exogenous MYC gene driven by a pGK promoter to allow cells to continue to proliferate if CTCF motif deletion is lethal (FIG. 9A). Sequence differences in the 3′ UTR allowed discrimination between the endogenous and exogenous MYC mRNAs. RNA analysis revealed that the CTCF binding site mutations in the MYC enhancer docking site caused a 70-80% reduction of endogenous MYC mRNA in K562, HCT-116, Jurkat and MCF7 cells (FIG. 3C). These results demonstrate that optimal expression of MYC in a spectrum of tumor cells is highly dependent upon the CTCF binding site sequence in the enhancer-docking site.

Loss of MYC Expression Upon Methylation of Enhancer-Docking Site

CTCF binding is abrogated when its sequence motif is methylated (Bell and Felsenfeld, 2000; Maurano et al., 2015), and the MYC enhancer-docking site occurs within a CpG island that is consistently hypomethylated in different tumor types (FIG. 10A). The recent development of tools that permit site-specific DNA methylation (Flavahan et al., 2016; Hark et al., 2000; Liu et al., 2016) suggested a means to disrupt MYC expression by methylation of the enhancer-docking site. To achieve targeted methylation, we created a construct to express a dCas9 fusion protein consisting of the catalytic domain of DNMT3A and the interacting domain of DMNT3L. This dCas9-DNMT3A-3L protein was targeted to the MYC enhancer-docking site using multiple guide RNAs that span the region (FIG. 4A, B). The targeting of dCas9-DNMT3A-3L resulted in robust local DNA methylation (FIG. 4C) and a 50-70% reduction in mRNA levels in HCT-116 and HEK293T cells (FIG. 4D). These results demonstrate that epigenetic editing of the enhancer-docking site can reduce MYC expression in multiple cell types.

MYC Enhancer-Docking Site in Normal Development and Differentiation

It seems unlikely that the MYC enhancer-docking site would have evolved to facilitate the development of diverse cancers, so we explored the possibility that this site might typically be employed during normal development and differentiation, when MYC expression contributes to normal cellular proliferation and different cell-type specific transcription factors establish the different cell states. Examination of enhancer and promoter-capture Hi-C data in a variety of normal cell types that express MYC (Javierre et al., 2016) revealed that cell-type specific enhancers do indeed loop to the MYC enhancer-docking site (FIG. 5A). The enhancer-docking site is hypomethylated in a broad spectrum of cell and tissue types (FIG. 5B, FIG. 10A,B), which would allow for CTCF binding at these sites. These results indicate that the MYC enhancer-docking site is used during normal development by cell-type specific enhancers to facilitate MYC expression and cellular proliferation. The levels of MYC transcripts in these normal cells, however, are considerably less than those found in tumor cells, where high levels of MYC expression produce oncogenic effects.

Enhancer-Docking Sites at Additional Genes with Prominent Roles in Cancer

Our initial analysis of putative enhancer-docking sites suggested that additional cancer-associated genes may be regulated in a manner similar to MYC (FIG. 1A). That analysis identified genes within insulated neighborhoods that have CTCF-bound sites at their promoters and that are expressed in multiple cell types. Among these genes were cancer associated genes such as VEGFA and RUNX1, but also developmentally active genes such as TGIF1 (FIG. 6A). These enhancer-docking sites tend to have sequence motifs that are highly conserved (FIG. 6B). These results show that multiple cancer-associated genes possess conserved enhancer-docking sites with properties similar to that described for MYC.

DISCUSSION

We describe here a class of human genes that utilize CTCF-CTCF interactions to connect different cell-type specific enhancers with a single promoter-proximal element that functions as a docking site for those enhancers. These CTCF-mediated enhancer-promoter interactions are generally nested within larger CTCF-mediated loops that function as insulated neighborhoods. At these genes with CTCF-mediated enhancer docking, the enhancers are often bound by CTCF in a cell-type specific fashion whereas the promoter-proximal enhancer-docking sites tend to be constitutively bound by CTCF.

The proto-oncogene MYC, which is controlled by different cell-type specific enhancers during development, is a prominent oncogene and an example of a gene regulated in this fashion. Many different human cancer cells acquire super-enhancers within the ˜3 MB MYC TAD/insulated neighborhood and we show here that these exploit a CTCF-mediated enhancer-docking mechanism to express MYC at oncogenic levels. Because tumor super-enhancers can encompass genomic regions as large as 200 kb, and CTCF occupies sites that occur on average every 10 kb, there is considerable opportunity for super-enhancers to adventitiously contain a CTCF-bound site, which in turn could serve to interact with an enhancer-docking site.

Additional genes with roles in cancer employ this CTCF-mediated enhancer-docking mechanism to engender interactions with tumor-specific enhancers. For example, at CSNK1A1, a drug target in AML tumor cells ({umlaut over (J)}r{dot over (a)}s et al., 2014), VEGFA, which is upregulated in many cancers (Goel and Mercurio, 2013), and RUNX1, a well-defined oncogene in AML (Deltcheva and Nimmo, 2017; Ito et al., 2015), the evidence indicates that super-enhancers in these cancer cells use a CTCF enhancer-docking site to interact with the oncogene. Thus, a CTCF-dependent enhancer-docking mechanism, which presumably facilitates interaction with different cell-specific enhancers during development, is exploited by cancer cells to dysregulate expression of prominent oncogenes.

MYC dysregulation is a hallmark of cancer (Bradner et al., 2017). The c-Myc TF is an attractive target for cancer therapy because of the role that excessive c-Myc levels play in a broad spectrum of aggressive cancers (Felsher and Bishop, 1999; Jain et al., 2002; Soucek et al., 2008, 2013), but direct pharmacologic inhibition of MYC remains an elusive challenge in drug discovery (Bradner et al., 2017). The MYC enhancer-docking site, and presumably those of other oncogenes, can be repressed by dCas9-DNMT-mediated DNA methylation. Oncogene enhancer-docking sites may thus represent a common vulnerability in multiple human cancers.

Materials and Methods:

Star Methods

Experimental Model and Subject Details

Cell Lines

K562 (female), MCF7 (female), HCT-116 (male), Jurkat (male) and HEK293T cells were purchased from ATCC (CCL-243, HTB-22, CCL-247, TIB-152, CRL-3216) and propagated according to ATCC guidelines in RPMI-1640 with GlutaMax (Life Technologies 61870-127) or DMEM, high glucose, pyruvate (Life Technologies 11995-073), supplemented with 10% fetal bovine serum (Sigma). Cells were maintained at 37° C. and 5% CO2.

Method Details

CRISPR/Cas9 Genome Editing

Genome editing was performed using CRISPR/Cas9 essentially as described (Ran et al., 2013; Shalem et al., 2014). The genomic sequences complementary to all guide RNAs are listed in Table S2

TABLE S2 FIG. 3 sequences gRNAs in pAW21 and pAW22 210-Δ.1 ACCGCCTGTCCTTCCCCCGC (SEQ ID NO: 6) 210-Δ.2 TTGGTTGCTCCCCGCGTTTG (SEQ ID NO: 7) 4C primers Fwd AGAGAGGCAGTCTGGTCATG (SEQ ID NO: 9) Rev CCAGTGTCTTGCTTTCAAAT (SEQ ID NO: 10) FIG. 4 sequences gRNA for CTCF motif deletion MYC_CTCF ATGATCTCTGCTGCCAGTAG (SEQ ID NO: 8) Primers for detection of endogenous MYC FWD AACCTCACAACCTTGGCTGA (SEQ ID NO: 11) REV TTCTTTTATGCCCAAAGTCCAA (SEQ ID NO: 12) Primers for detection of exogenous MYC FWD TGATCCTAGCAGAAGCACAGG (SEQ ID NO: 13) REV TGGACGAGCTGTTACAAGAGC (SEQ ID NO: 14) Primers for detection of GAPDH FWD TGCACCACCAACTGCTTAGC (SEQ ID NO: 15) REV GGCATGGACTGTGGTCATGAG (SEQ ID NO: 16) FIG. 5 sequences sgRNAs in pJS-DNMT3A-3L and pLentiGuide Puro GCCTGGATGTCAACGAGGGC (SEQ ID NO: 1) GCGGGTGCTGCCCAGAGAGG (SEQ ID NO: 2) GCAAAATCCAGCATAGCGAT (SEQ ID NO: 3) CTATTCAACCGCATAAGAGA (SEQ ID NO: 4) CGCTGAGCTGCAAACTCAAC (SEQ ID NO: 5) Primers for Bisulfite PCR FWD AAGGAGGTGGYTGGAAAYTT (SEQ ID NO: 17) REV TCCCTCCACCACCTCCAAAA (SEQ ID NO: 18) FIG. 9 sequences Primers for genotyping FWD TCTGAACCACTTTTTCCTCCA (SEQ ID NO: 19) REV ACTGGCAGCAGAGATCATCG (SEQ ID NO: 20)

For generation of Myc-cover clones, target-specific oligonucleotides were cloned into the pX330 plasmid which carries a U6 promoter, chimeric guide RNA, and a codon-optimized version of Cas9. pX330 was a kind gift of F. Zhang (Cong et al., 2013) (Addgene: 42230). For the generation of Myc-cover line clones, 2 million cells were transfected with 10 ug of DNA with 50 uL of 1 mg/ml PEI and sorted for presence of GFP after 2 days. Individual cells were then propagated in to clonal lines.

For the 210-Δ experiments (FIG. 3), target specific oligonucleotides were cloned into a plasmid containing the chimeric RNA, a doxycycline inducible H1 promoter, TetR, and a selectable marker (pAW 21 Addgene 85673 or pAW 22 Addgene 85674). Three separate viruses were produced containing pAW21::up guide, pAW22::down guide, and pLentiCas9-blast (Addgene: 52962, a kind gift of F. Zhang (Sanjana et al., 2014)). Stable cell lines were generated (see section on virus production and cell line generation) and then genome editing was induced by the addition of doxycycline (Sigma Aldrich D9891) at 1 ug/mL. Cells were induced for 72 hours and every 24 hours fresh doxycycline was spiked in.

Virus Production and Generation of Cell Lines

For virus production, HEK293T cells grown to 50-75% confluency on a 15 cm dish and then transfected with 15 μg plasmid of interest, 11.25 μg psPAX (Addgene 12260), and 3.75 μg pMD2.G (Addgene 12259). psPAX and pMD2.G were kind gifts of Didier Trono. After 12 hours, media was replaced. Viral supernatant was collected 24 hours after media replacement (36 hrs post transfection) and fresh media was added. Viral supernatant was collected again 48 hours after the media replacement (60 hours post transfection). Viral supernatant was cleared of cells by either centrifugation at 500×g for 10 minutes or filtration through a 0.45-micron filter. The virus was concentrated with Lenti-X concentrator (Clonetech 631231) per manufacturers' instruction. Concentrated virus was resuspended in either DMEM or RPMI (depending on the cell line being infected) and added to 5 million cells in the presence of polybrene (Millipore TR-1003) at 8 ug/mL. After 24 hours, viral media was removed and fresh media containing drug was added. Drug concentrations are as follows: Puromycin (Thermo A1113802) (2 ug/mL), Geneticin (Thermo 10131027) (800 ug/mL), Blasticidin (Invivogen ant-b1-1) (10 ug/mL). Cells were selected until all cells on non-transduced plates died. The viral plasmid containing pGK-MYC-tdTomato was deposited on Addgene (Plasmid #85675).

RNA Isolation and Quantitative RT-PCR

RNA was isolated using the RNeasy, RNeasy plus or AllPrep kit (QIAGEN 74004, 80204) and reverse transcribed using oligo-dT primers (Promega C1101) and SuperScript III reverse transcriptase (Thermo 18080093) according to the manufacturers' instructions. Quantitative real-time PCR was performed on a 7000 AB Detection System using Taqman probes for MYC (Hs00153408_m1) and GAPDH (Hs02758991_g1) in conjunction with Taqman 2× master mix (Thermo 4304437). For detection of endogenous MYC only in experiments utilizing the MYC cover, primers specific to the endogenous copy of MYC (Table S2) were designed against a MYC 3′ UTR region not present in the cover construct and qPCR was conducted with SYBR green PCR master mix (Thermo 4309155).

ChIP-seq

ChIP was performed as described in (Lee et al., 2006) with a few adaptations. ˜30 million K562 cells were crosslinked for 10 min at room temperature by the addition of one-tenth of the volume of 11% formaldehyde solution (11% formaldehyde, 50 mM HEPES pH 7.3, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0) to the growth media followed by 5 min quenching with 125 mM glycine. Cells were washed twice with PBS, then the supernatant was aspirated and the cell pellet was flash frozen at −80 C. 100 μl of Protein G Dynabeads (Thermo 10003D) were blocked with 0.5% BSA (w/v) in PBS. Magnetic beads were bound with 40 μl of anti-CTCF antibody (Millipore 07-729). Nuclei were isolated as previously described (Lee et al., 2006), and sonicated in lysis buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH8.0, 0.1% SDS, and 1% Triton X-100) on a Misonix 3000 sonicator for 5 cycles at 30s each on ice (18-21 W) with 60 s on ice between cycles. Sonicated lysates were cleared once by centrifugation and incubated overnight at 4° C. with magnetic beads bound with antibody to enrich for DNA fragments bound by the indicated factor. Beads were washed with wash buffer A (50 mM HEPES-KOH pH7.9, 140 mM NaCl, 1 mM EDTA pH 8.0, 0.1% Na-Deoxycholate, 1% Triton X-100, 0.1% SDS), B (50 mM HEPES-KOH pH7.9, 500 mM NaCl, 1 mM EDTA pH 8.0, 0.1% Na-Deoxycholate, 1% Triton X-100, 0.1% SDS), C (20 mM Tris-HCl pH8.0, 250 mMLiCl, 1 mM EDTA pH 8.0, 0.5% Na-Deoxycholate, 0.5% IGEPAL C-630 0.1% SDS) and D (TE with 50 mM NaCl) sequentially. DNA was eluted in elution buffer (50 mM Tris-HCL pH 8.0, 10 mM EDTA, 1% SDS). Cross-links were reversed overnight at 65° C. RNA and protein were digested using RNase A and Proteinase K, respectively and DNA was purified with phenol chloroform extraction and ethanol precipitation. Purified ChIP DNA was used to prepare Illumina multiplexed sequencing libraries. Libraries for Illumina sequencing were prepared following the Illumina TruSeq DNA Sample Preparation v2 kit. Amplified libraries were size-selected using a 2% gel cassette in the Pippin Prep system from Sage Science set to capture fragments between 200 and 400 bp. Libraries were quantified by qPCR using the KAPA Biosystems Illumina Library Quantification kit according to kit protocols. Libraries were sequenced on the Illumina HiSeq 2500 for 40 bases in single read mode.

4C-seq

A modified version of 4C-seq (van de Werken et al., 2012a, 2012b) was developed. The major change was the ligation is performed in intact nuclei (in situ). This change was incorporated because previous work has noted that in situ ligation dramatically decreases the rate of chimeric ligations and background interactions (Nagano et al., 2015; Rao et al., 2014).

Approximately 5 million K562 cells were resuspended in 5 mL 10% FBS/PBS. 5 mL of 4% formaldehyde in 10% FBS/PBS was added and cells were crosslinked for 10 minutes while rotating at room temperature. Glycine was added to a final concentration of 0.125 M and cells were centrifuged at 300×g for 5 minutes. Cells were washed twice with PBS, transferred to an eppendorf tube, snap frozen and stored at −80. Pellets were gently resuspended in Hi-C lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% Igepal) with 1× protease inhibitors (Roche, 11697498001). Cells were incubated on ice for 30 minutes then washed once with 500 uL of ice-cold Hi-C lysis buffer with no protease inhibitors. Pellets were resuspended in 50 uL of 0.5% SDS and incubated at 62° C. for 7 minutes. 145 uL of water and 25 uL of 10% Triton X-100 were added and tubes incubated at 37° C. for 15 minutes. 25 uL of 10× New England Biolabs CutSmart buffer and 200 units of NlaIII (NEB R0125L) enzyme were added and the chromatin was digested for four hours at 37 degrees in a thermomixer at 500 RPM. 200 additional units of NlaIII was spiked in and digest continued for 12 hours. Then, 200 additional units of NlaIII was spiked in and digest continued for four more hours. Restriction enzyme was inactivated by heating to 62° C. for 20 minutes while shaking at 500 rpm. Proximity ligation was performed in a total of 1200 uL with 2000 units of T4 DNA ligase (NEB M0202M) for six hours at room temperature. After ligation samples were spun down for 5 minutes at 2500 g and resuspended in 300 uL 10 mM Tris-HCl, 1% SDS and 0.5 mM NaCl with 1000 units of Proteinase K. Samples were reversed cross-linked overnight at 68° C.

Samples were then phenol-chloroform extracted and ethanol precipitated and the second digestion was performed overnight in 450 uL with 50 units of CviQI (NEB R0639L). Samples were phenol-chloroform extracted and ethanol precipitated and the second ligation was performed in 14 mL total with 6700 units of T4 DNA ligase (NEB 0202M) at 16° C. overnight. Samples were ethanol precipitated, resuspended in 500 uL Qiagen EB buffer, and purified with a Qiagen PCR kit.

The concentration was measured with a Nanodrop and PCR amplification was performed with 16 50 uL PCR reactions using Roche Expand Long Template polymerase (Roche 11759060001). Reaction conditions are as follows: 11.2 uL Roche Expand Long Template Polymerase, 80 uL of 10×Roche Buffer 1, 16 uL of 10 mM dNTPs (Promega PAU1515), 112 uL of 10 uM forward primer, 112 uL of 10 uM reverse primer (Table S2), 200 ng template, and milli-q water till 800 uL total. Reactions were mixed and then distributed into 16 50 uL reactions for amplification. Cycling conditions were a “Touchdown PCR” based on reports that this decreases non-specific amplification of 4 C libraries (Ghavi-Helm et al., 2014). The conditions are: 2′ 94° C., 10″ 94° C., 1′ 63° C., 3′ 68° C., repeat steps 2-4 but decrease annealing temperature by one degree, until 53° C. is reached at which point reaction is cycled an additional 15 times at 53° C., after 25 total cycles are performed the reaction is held for 5′ at 68° C. and then 4° C. Libraries were cleaned-up using a Roche PCR purification kit (Roche 11732676001) using 4 columns per library. Reactions were then further purified with Ampure XP beads (Agencourt A63882) with a 1:1 ratio of bead solution to library following the manufactures instructions. Samples were then quantified with Qubit and the KAPA Biosystems Illumina Library Quantification kit according to kit protocols. Libraries were sequenced on the Illumina HiSeq 2500 for 40 bases in single read mode.

HiChIP

HiChIP was performed essentially as described (Mumbach et al., 2016). 10 million HCT116 cells were crosslinked for 10 min at room temperature by the addition of one-tenth of the volume of 11% formaldehyde solution (11% formaldehyde, 50 mM HEPES pH 7.3, 100 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0) to the growth media followed by 5 min quenching with 125 mM glycine. Cells were washed twice with PBS, then the supernatant was aspirated and the cell pellet was flash frozen in liquid nitrogen. Frozen crosslinked cells were stored at −80° C.

The crosslinked pellets were thawed on ice, resuspended in 500 μL of ice-cold Hi-C Lysis Buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP-40 with protease inhibitors), and rotated at 4° C. for 30 minutes. Nuclei were spun down at 2500 rcf for 5 minutes at 4° C., and washed once with 500 μL of ice-cold Hi-C Lysis Buffer. Supernatant was removed, and the pellet was resuspended in 100 μL of 0.5% SDS. Nuclei were incubated at 62° C. for 7 minutes, and SDS was quenched by addition of 285 μL of H₂O and 50 μL of 10% Triton X-100 for 15 minutes at 37° C. After the addition of 50 μL of 10×NEB Buffer 2 and 400 U of MboI restriction enzyme (NEB, R0147), chromatin was digested overnight at 37° C. The following day, the MboI enzyme was inactivated by incubating the nuclei at 62° C. for 20 minutes.

To fill in the restriction fragment overhangs and mark the DNA ends with biotin, the following was added: 37.5 μL 0.4 mM biotin-ATP (19524-016, Invitrogen) 1.5 μL of 10 mM dCTP (N0441S, NEB), 1.5 μL of 10 mM dTTP (N0443S, NEB), 1.5 μL of 10 mM dGTP (N0442S, NEB), 10 μL of 5 U/μL DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210), and the tubes were incubated at 37° C. for 1 hour with rotation. Next, the following mix was added for the proximity ligation step: 150 μL of 10×NEB T4 DNA ligase buffer with 10 mM ATP (NEB, B0202), 125 μL 10% Triton X-100, 3 μL 50 mg/mL BSA, 10 μL 400 U/μL T4 DNA Ligase (NEB, M0202), 660 μL H2O, and the nuclei suspension was incubated at room temperature for 6 hours with rotation. Nuclei were pelleted at 2500 rcf for 5 minutes and supernatant was removed.

Pellets were resuspended in 880 μL in Nuclear Lysis Buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS with protease inhibitors), and the lysates were sonicated on a Covaris S220 instrument using the following parameters: Fill Level 10, Duty Cycle 5, PIP 140, Cycles/Burst 200, for 4 minutes. Sonicated lysates were spun down at 16100 rcf for 15 minutes at 4° C., and the supernatant was transferred to a fresh tube. The supernatant was split into two Eppendorf tubes (about 400 μL of lysate in each), and 800 μL of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 7.5, 167 mM NaCl) was added to each tube. 60 μL of Protein G beads (Life Technologies) were washed in ChIP dilution buffer, resuspended in 100 μL ChIP dilution buffer and 50 μL were added to each of the two tubes of lysates. Tubes were then rotated for 1 hour at 4° C. to preclear the lysates. Dynabeads were separated on a magnetic stand, and the supernatant was moved to a fresh tube. 3.5 μg of SMC1A antibody (Bethyl A300-OSSA) were added to each tube, and tubes were incubated at 4° C. overnight with rotation. The next day, 60 μL of Protein G beads were washed ChIP Dilution Buffer, resuspended in 100 μL ChIP Dilution Buffer, and 50 μL was added to each sample tube. Samples were then incubated for 2 hours at 4° C. with rotation. Beads were washed twice with Low Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 7.5, 150 mM NaCl), twice with High Salt Wash Buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 7.5, 500 mM NaCl), twice with LiCl buffer (10 mM Tris pH 7.5, 250 mM LiCl, 1% NP-40, 1% Sodium-deoxycholate, 1 mM EDTA). Beads were then resuspended in 100 μL of DNA Elution Buffer (50 mM NaHCO3, 1% SDS), incubated for 10 minutes at room temperature with rotation and 3 minutes at 37° C. with shaking. Beads were separated on a magnetic stand, and supernatant was transferred to a fresh tube. Beads were then mixed with another 100 μL of DNA Elution Buffer, incubated for 10 minutes at room temperature with rotation and 3 minutes at 37° C. with shaking. Beads were separated on a magnetic stand, and supernatant was combined with the previous round of supernatant. 10 μL of Proteinase K (20 mg/ml) was added to each sample and samples were incubated at 55° C. for 45 minutes with shaking. Temperature was then increased to 67° C., and samples were incubated for 1.5 hours with shaking. Samples were purified on a Zymo column (Zymo Research).

Fragmentation of the ChIP DNA was performed using the Tn5 transposase (Illumina). First, 5 μL of Streptavidin M-280 magnetic beads were washed with Tween Wash Buffer (5 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20), resuspended in 10 μL of Binding Buffer (10 mM, Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl), and added to the samples. Samples were then rotated for 15 minutes at room temperature. Beads were separated on a magnet, and supernatant was discarded. Beads were washed twice with 500 μL of Tween Wash Buffer and incubated at 55° C. for 2 minutes shaking. Beads were then washed with 100 μL of 1× TD Buffer (Nextera DNA sample preparation kit, FC-121-1030, Illumina), and tagmented using the Nextera DNA sample preparation kit (FC-121-1030, Illumina) 0.5 μL TDE1 enzyme was used to tagment 10 ng of ChIP DNA (quantified after the previous Zymo column purification). Tagmentation was performed for 10 minutes at 55° C. with shaking. Beads were then separated on a magnet, and supernatant was discarded. Beads were washed with 50 mM EDTA at 50° C. for 30 minutes, and twice with 50 mM EDTA at 50° C. for 3 minutes. Beads were then washed twice in Tween Wash Buffer at 55° C. for 2 minutes, and once with 10 mM Tris for 1 minute at room temperature. The tagmented library still bound to the beads was amplified by 12 cycles of PCR using the Nextera DNA sample preparation kit. The library was then purified on a Zymo column, size-selected (300-700 bp) using AMPure beads (Agencourt) per manufacturers' instructions, and sequenced 100×100 on an Illumina Hi-Seq 2500.

Targeted Methylation and Bisulfite Sequencing.

To perform targeted methylation, HCT-116 cells or HEK293T were transfected with a dCas9-DNMT3A-3L construct with or without guides. To generate the dCas9-DNMT3A-3L construct, dCas9 was isolated from pSQL1658 (Addgene: 51023) by PCR. Cas9 was removed from pX330-Cas9 (Addgene: 42230) by Agel and EcoRI restriction digest. dCas9 was inserted into pX330 to create pX330-dCas9. DNMT3A-3L carrier plasmid was a generous gift from the Jeltsch lab (Siddique et al., 2013). DNMT3A-3L was cloned into pX330-dCas9 using Pmel and Ascl sites to create pX330-dCas9-DNMT3A-3L (no guides). Guide RNAs were added to pX330-dCas9-DNMT3A-3L by digesting pX330-dCas9-DNMT3A-3L with Bbsl followed by ligation of annealed oligos (Table S2) to create pX330-dCas9-DNMT3A-3L-guide with three different guides. An additional plasmid containing two guides, lentiGuide-Puro-double_guide, was also generated. Double guide containing gBlock (individual guide sequences Table S2) was cloned into lentiGuide-Puro (Addgene: 52963) as described (Vidigal and Ventura, 2015) to create lentiGuide-Puro-double_guide. Two hundred fifty thousand HCT-116 or HEK293T cells were transfected with either 750 ng of pX330-dCas9-DNMT3A-3L (no guides) (Addgene: 85701), 250 ng lentiGuide-Puro, and 5 uL of lmg/ml PEI, or 250 ng of pX330-dCas9-DNMT3A-3L-guide1, 250 ng of pX330-dCas9-DNMT3A-3L-guide2, 250 ng of pX330-dCas9-DNMT3A-3L-guide3, 250 ng lentiGuide-Puro-double_guide, and 5 uL of 1 mg/ml PEI, and harvested after two days. HCT-116 cells were harvested and sorted for GFP presence after two days, HEK293T cells were harvested without cell sorting.

To detect methylation, 2 ug of gDNA from HCT-116 or HEK293T cells transfected with dCas9-DNMT3A-3L or dCas9-DNMT3A-3L plus guides were bisulfite converted using the EpiTect Bisulfite Kit (QIAGEN 59104). Converted gDNA was eluted in 20 uL H2O. Converted gDNA was PCR amplified with EpiMark® Hot Start Taq DNA Polymerase (NEB M0490) using 3 uL of converted gDNA as template and locus specific primers (Table S2). PCR was carried out as follows: 95° C. for 30 sec; 95° C. for 20 sec; 52° C. for 30 sec; 68° C. for 30 sec; repeat steps 2-4 45×; 68° C. for 5 min; Hold 4° C. Resultant amplicons were cleaned up using QIAquick PCR Purification Kit (QIAGEN 28106) and eluted in 20 ul H2O. Clean amplicons (3 uL) were subcloned using the pGEM-T Easy vector system (Promega A1360) and transformed into DH5α competent cells. Individual colonies were then picked, and colony PCR was carried out using GoTaq Green Master Mix (Promega M712) with the same primers originally used for the converted gDNA amplification. Colony PCR was carried out as follows: 95° C. for 2 min; 95° C. for 45 sec; 51° C. for 45 sec; 72° C. for 45 sec; repeat steps 2-4 29×; 72° C. 5 min; Hold 4° C. Resultant amplicons were then Sanger sequenced, and CpG methylation was detected as CpG sequences that were not converted to TpG. All converted DNA analyzed had >95% bisulfite conversion rate.

Hi-C Visualization

Hi-C datasets were visualized using the 3D Genome browser at http://www.3dgenome.org.

Visualization of ChIA-PET Interactions on the WashU Genome Browser

The output of origami was visualized in the WashU genome browser by converting the output of origami into a WashU compatible format using origami-conversion.

Topologically Associating Domain (TAD) Calls

TAD calls were taken from the TAD calls in (Dixon et al., 2012) from the H1 human embryonic cell line.

Quantification and Statistical Analysis

ChIP-Seq Data Analysis

ChIP-Seq datasets were generated for this study as well as collated from previous studies (Table S3), and were aligned using Bowtie (version 0.12.2) (44) to the human genome (build hg19, GRCh37) with parameter −k 1 −m 1 −n 2. We used the MACS version 1.4.2 (model-based analysis of ChIP-seq) (45) peak finding algorithm to identify regions of ChIP-seq enrichment over input DNA control with the parameter “--no-model--keep-dup=auto”. A p-value threshold of enrichment of 1e-09 was used. UCSC Genome Browser tracks were generated using MACS wiggle outputs with parameters “-w -S -space=50”. The browser snapshots of the ChIP-Seq binding profiles displayed throughout the study use read per kilobase per million mapped reads dimension (rpm/bp) on the y-axis.

TABLE S3 Datasets and their reference numbers used in this study ChIP-seq Origin Factor GEO HCT-116 H3K27Ac GSM945853 HCT-116 Input GSM749774 GM12878 H3K27Ac GSM733771 GM12878 Input GSM733742 MCF-7 H3K27Ac GSM946850 MCF-7 Input GSM945859 Pane1 H3K27Ac GSM818826 Pane1 Input GSM818828 LnCAP H3K27Ac GSM686937 LnCAP Input GSM686947 u87 H3K27Ac GSM894065 u87 Input GSM894096 ccRCC H3K27Ac GSM1960256 ccRCC Input GSM1960260 Jurkat H3K27Ac GSM1224780 Jurkat Input GSM569086 K562 H3K27Ac GSM733656 K562 Input GSM733780 HEK293T H3K27Ac GSE92879 HEK293T Input GSM1910999 GM12878 CTCF GSM749706 Jurkat CTCF GSM1689152 K562 CTCF GSE92879 K562 Input GSE92879 ΔK562 CTCF GSE92879 HEK293T CTCF GSM749668 HCT-116 CTCF GSM1010903 MCF7 CTCF GSM1022658 MCF7 Input GSM945859 Dnd41 CTCF GSM1003464 GP5D CTCF GSM1240813 LoVo CTCF GSM1239390 HeLa CTCF GSM749729 HeLa Input SRX1097060 Caco2 CTCF GSM749748 GM12878 CTCF GSM749706 GM12875 CTCF GSM749764 A549 CTCF GSM1003606 HepG2 CTCF GSM803486 MM1.S CTCF GSM1070125 Mouse CTCF GSM747536 Opossum CTCF E-MTAB-437 Dog CTCF E-MTAB-437 Rhesus CTCF E-MTAB-437 Chicken CTCF GSE51846 DNA interaction data Origin Factor Technique GEO MCF7 PolII ChIA-PET GSE33664 MCF7 CTCF ChIA-PET GSM970215 K562 RAD21 ChIA-PET GSM1436264 Jurkat SMC1 ChIA-PET GSE68977 HCT-116 SMC1 HiChIP GSE92879

Identification of Enhancers and Super-Enhancers

Enhancers and super-enhancers were identified using H3K27Ac ChIP-seq data as previously described (Hnisz et al., 2013). Briefly, enhancers were defined as H3K27Ac ChIP-Seq peaks identified using MACS. To identify super-enhancers, the H3K27Ac ChIP-Seq peaks (i.e. enhancers) were stitched together if they were within 12.5 kb, and the stitched enhancers were ranked by their ChIP-seq read signal of H3K27Ac, using the ROSE algorithm (https://bitbucket.org/young_computation/rose) (Lovén et al., 2013). ROSE separates super-enhancers from typical enhancers by identifying an inflection point of H3K27ac signal vs. enhancer rank (Hnisz et al., 2013; Lovén et al., 2013).

Super-enhancers across cancer types and their H3K27Ac ChIP-seq densities Chrom start stop from H3K27Ac input /input chr8 128901634 128963789 Brain-u87 46516.8 10547.7 4.410136 chr8 129165089 129210112 Brain-u87 33222.47 7550.357 4.400119 chr8 128795447 128824370 Breast-MCF7 29790.69 8491.793 3.508174 chr8 128860824 128884150 Breast-MCF7 31550.75 9038.825 3.490581 chr8 129147244 129191727 Breast-MCF7 48980.23 13696.32 3.576161 chr8 128901900 128941452 CML-K562 54601.54 5758.771 9.481456 chr8 129046437 129091141 CML-K562 66448.03 6437.376 10.32222 chr8 130564436 130604313 CML-K562 54930.57 7141.971 7.691234 chr8 130690815 130723494 CML-K562 44322.53 5202.497 8.519472 chr8 128957467 128984610 CML-K562 29181.44 4166.451 7.003909 chr8 128744519 128756323 Colorectal-HCT116 40916.21 1541.602 26.54135 chr8 128208736 128320580 Colorectal-HCT116 190906.5 18756.24 10.17829 chr8 128190704 128239822 DLBCL-GM12878 115388 3281.082 35.16766 chr8 128298980 128321170 DLBCL-GM12878 61690.42 1835.113 33.61669 chr8 129594445 129618316 Liver-HEPG2 62107.57 23615.58 2.62994 chr8 128805716 128945203 Pancreatic-panc1 231869.2 13627.88 17.01433 chr8 128805846 128836987 PrimaryKidney-ccRCCII 15523.79 3512.705 4.419326 chr8 128857246 128942648 PrimaryKidney-ccRCCII 65699.76 13698.48 4.796135 chr8 128746055 128755790 Prostate-LnCAP 25723.76 1478.747 17.39566 chr8 128810486 128990391 Prostate-LnCAP 189655.9 24664.98 7.689278 chr8 129942458 130013116 T-ALL-Jurkat 43984.61 8478.96 5.1875 chr8 130030058 130115864 T-ALL-Jurkat 81018.03 11266.33 7.191165 chr8 130148897 130290103 T-ALL-Jurkat 190811.7 19359.34 9.856309 chr8 130345866 130473031 T-ALL-Jurkat 156082.3 16048.22 9.725832 chr8 130540472 130572787 T-ALL-Jurkat 31678.39 4262.349 7.432146

4C Analysis

The 4C-seq samples were first processed by removing their associated read primer sequences from the 5′ end of each FASTQ read. To improve mapping efficiency of the trimmed reads by making the read longer, the restriction enzyme digest site was kept on the trimmed read. After trimming the reads, the reads were mapped using bowtie with options -k 1-m 1 against the hg19 genome assembly. All unmapped or repetitively mapping reads were discarded from further analysis. The hg19 genome was then “digested” in silico according to the restriction enzyme pair used for that sample to identify all the fragments that could be generated by a 4C experiment given a restriction enzyme pair. All mapped reads were assigned to their corresponding fragment based on where they mapped to the genome. The digestion of a sample in a 4C experiment creates a series of “blind” and “non-blind” fragments as described (van de Werken et al., 2012b). In a perfect experiment, we should have only observed reads at non-blind fragments, and reads at blind fragments exhibit a much higher experimental variability than non-blind fragments, so we only used the reads from non-blind fragments for further analysis. To normalize the distribution of different samples, we quantile normalized all non-blind fragments in each sample together. If no reads were detected at a non-blind fragment for a given sample when reads were detected in at least one other sample, we assigned a “0” to that non-blind fragment for the sample(s) missing reads. After normalization, we then smoothed the normalized profile of each sample using a 10 kb running mean at 1000 bp steps across the genome. After smoothing, for each condition we combined the replicates of a condition by taking the mean signal of each bin across all replicates of the condition.

HiChIP Data Analysis

The HiChIP samples were processed by removing their associated read primer sequences from the 5′ end of each FASTQ read. Read pairs were separated and separate reads were mapped using bowtie with options -k 1-m 1 against the hg19 genome assembly. Al unmapped or repetitively mapping read were discarded from further analysis. The hg19 genome was then divided in 50 kb bins and reads were joined back together in pairs (Paired End Tag PET). For every pair of bins the number of PETS joining them was then calculated. These data were then further analyzed by the ORIGAMI pipeline to identify significant bin to bin interaction pairs.

ChIA-PET Data Analysis and ORIGAMI Description

We development a new software pipeline and analytical method called origami to process ChIA-PET. The software and releases can be found at https://github.com/younglab/origami using version alpha20160828. Each ChIA-PET data sets was processed as follows: the reads were first trimmed and aligned using origami-alignment, which trims the ChIA-PET linker if present and aligns trimmed PETs. PETs not having a linker were discarded from further analysis. Each end of a PET with a linker sequence were separately mapped to the hg19 genome assembly using bowtie with the following options: -v 1-k 1-m 1. After alignment, the separated PETs were re-paired in the final BAM output. After repairing, all duplicated PETs within the data were removed, since these were believed to be PCR duplicates. Peaks were called on the re-paired ChIA-PET reads using MACS1 v1.4.2 with the following parameters: --nolambda -nomodel -p 1e-9. The ChIA-PET data analyzed with their corresponding linker sequence can be found in Table S4.

TABLE S4 Rad21 ChIA-PET interactions identified using the Mango pipeline within chr8:127100000-131525000 Origami Anchor 1 Anchor 2 Posterior chr start end chr start end score chr8 100023513 100026504 chr8 135844278 135845179 0 chr8 101552284 101554003 chr8 146227224 146228981 0 chr8 101575720 101577906 chr8 144804210 144805137 0 chr8 101776000 101776619 chr8 129201051 129204117 0 chr8 101993048 101993904 chr8 144613630 144615202 0.044 chr8 102138237 102139789 chr8 130572932 130573977 0 chr8 102148782 102152241 chr8 144648850 144650112 0.045 chr8 102215344 102218586 chr8 131662983 131664104 0 chr8 103079364 103080681 chr8 143528588 143529404 0.001 chr8 103579789 103581463 chr8 130690599 130698597 0 chr8 103754509 103755759 chr8 139779408 139780700 0 chr8 103822007 103824271 chr8 129074007 129074657 0.069 chr8 103874149 103877683 chr8 143556624 143557688 0 chr8 10405232 10406385 chr8 130458049 130460184 0 chr8 10447038 10451285 chr8 145687898 145694087 0 chr8 10548811 10549484 chr8 142440979 142442237 0 chr8 106103943 106106051 chr8 130708318 130711352 0.001 chr8 106328459 106332780 chr8 145747174 145749210 0 chr8 107603518 107604902 chr8 130690599 130698597 0.078 chr8 11266584 11268410 chr8 144976258 144977630 0 chr8 11424252 11425502 chr8 144818191 144819803 0 chr8 116439654 116440634 chr8 130586622 130588918 0.071 chr8 11758993 11760861 chr8 144814755 144816677 0 chr8 117635580 117636837 chr8 146124945 146128287 0 chr8 123687920 123688890 chr8 128863351 128864822 0 chr8 123689226 123691973 chr8 142396269 142398926 0 chr8 123701700 123702600 chr8 145668976 145670660 0 chr8 124048990 124051516 chr8 131539617 131541377 0 chr8 124052796 124055142 chr8 142309091 142310265 0 chr8 124166473 124167842 chr8 130690599 130698597 0 chr8 124427965 124430186 chr8 144512427 144514351 0 chr8 124775023 124781216 chr8 135898207 135899420 0 chr8 124933901 124936146 chr8 131687123 131688649 0.059 chr8 124933901 124936146 chr8 135643306 135644060 0 chr8 124936563 124937824 chr8 130568321 130570573 0 chr8 125049074 125050755 chr8 144766236 144767123 0 chr8 125576122 125577245 chr8 127821728 127822652 0 chr8 125900120 125901746 chr8 127312430 127314227 0 chr8 125968009 125969031 chr8 141645298 141648346 0 chr8 12611291 12611897 chr8 144678737 144680359 0 chr8 126438288 126439362 chr8 146029960 146030748 0 chr8 126440793 126445829 chr8 128585179 128586353 0 chr8 126440793 126445829 chr8 145022340 145027313 0 chr8 126714207 126715689 chr8 127395261 127396692 0 chr8 126714207 126715689 chr8 127821728 127822652 0 chr8 126714207 126715689 chr8 127836281 127837654 0 chr8 126741377 126742014 chr8 127395261 127396692 0 chr8 126923847 126924704 chr8 127312430 127314227 0 chr8 126923847 126924704 chr8 127395261 127396692 0 chr8 12697849 12699125 chr8 143807971 143809086 0 chr8 127312430 127314227 chr8 127314423 127315266 1 chr8 127312430 127314227 chr8 127395261 127396692 1 chr8 127312430 127314227 chr8 127821728 127822652 0.803 chr8 127314423 127315266 chr8 127395261 127396692 0.034 chr8 127836281 127837654 chr8 128190325 128191348 0 chr8 127836281 127837654 chr8 128745001 128752294 0 chr8 127836281 127837654 chr8 129201051 129204117 0 chr8 127881465 127882077 chr8 127888216 127890235 0 chr8 127881465 127882077 chr8 128755105 128756028 0.022 chr8 127888216 127890235 chr8 130598258 130600266 0 chr8 127888216 127890235 chr8 130690599 130698597 0.439 chr8 127888216 127890235 chr8 130708318 130711352 0 chr8 128190325 128191348 chr8 128412665 128413632 0 chr8 128190325 128191348 chr8 130698611 130700895 0 chr8 128190325 128191348 chr8 130708318 130711352 0 chr8 128412665 128413632 chr8 128772026 128773570 0 chr8 128412665 128413632 chr8 130546973 130552572 0 chr8 128412665 128413632 chr8 130737602 130739686 0 chr8 128585179 128586353 chr8 128737215 128739057 0.001 chr8 128585179 128586353 chr8 128740727 128741574 0.001 chr8 128585179 128586353 chr8 128745001 128752294 0 chr8 128585179 128586353 chr8 129060227 129063805 0.033 chr8 128585179 128586353 chr8 129870968 129871979 0 chr8 128585179 128586353 chr8 130546973 130552572 0 chr8 128737215 128739057 chr8 128739537 128740494 1 chr8 128737215 128739057 chr8 128740727 128741574 0.942 chr8 128737215 128739057 chr8 128745001 128752294 0.999 chr8 128737215 128739057 chr8 129201051 129204117 0 chr8 128737215 128739057 chr8 129209019 129210639 0 chr8 128737215 128739057 chr8 129323874 129325067 0.021 chr8 128737215 128739057 chr8 129665003 129666346 0.82 chr8 128737215 128739057 chr8 129870968 129871979 0 chr8 128737215 128739057 chr8 130690599 130698597 0.04 chr8 128737215 128739057 chr8 130698611 130700895 0 chr8 128737215 128739057 chr8 130737602 130739686 0 chr8 128737215 128739057 chr8 145022340 145027313 0 chr8 128739537 128740494 chr8 128740727 128741574 0 chr8 128739537 128740494 chr8 128745001 128752294 0.073 chr8 128739537 128740494 chr8 128772026 128773570 0 chr8 128739537 128740494 chr8 129201051 129204117 0 chr8 128739537 128740494 chr8 130690599 130698597 0 chr8 128740727 128741574 chr8 128745001 128752294 1 chr8 128740727 128741574 chr8 130605506 130606759 0 chr8 128745001 128752294 chr8 128752908 128753569 0.976 chr8 128745001 128752294 chr8 128772026 128773570 0.985 chr8 128745001 128752294 chr8 128812838 128813544 0 chr8 128745001 128752294 chr8 128830032 128831239 1 chr8 128745001 128752294 chr8 128863351 128864822 0.022 chr8 128745001 128752294 chr8 128871531 128872830 0.762 chr8 128745001 128752294 chr8 128906465 128907622 0.96 chr8 128745001 128752294 chr8 128910320 128912382 0.248 chr8 128745001 128752294 chr8 128922538 128924783 0.959 chr8 128745001 128752294 chr8 128926524 128927220 0 chr8 128745001 128752294 chr8 128971685 128973318 0.224 chr8 128745001 128752294 chr8 128973335 128974261 0.039 chr8 128745001 128752294 chr8 128979417 128982624 0.021 chr8 128745001 128752294 chr8 128982695 128983816 0.03 chr8 128745001 128752294 chr8 129005065 129006002 0.284 chr8 128745001 128752294 chr8 129056676 129058361 0.019 chr8 128745001 128752294 chr8 129179729 129180447 0 chr8 128745001 128752294 chr8 129188678 129190475 0 chr8 128745001 128752294 chr8 129196702 129197452 0.03 chr8 128745001 128752294 chr8 129201051 129204117 1 chr8 128745001 128752294 chr8 129205265 129206000 0 chr8 128745001 128752294 chr8 129209019 129210639 0.033 chr8 128745001 128752294 chr8 129334525 129335739 0.993 chr8 128745001 128752294 chr8 129665003 129666346 0.04 chr8 128745001 128752294 chr8 129870968 129871979 0 chr8 128745001 128752294 chr8 130046989 130047842 0 chr8 128745001 128752294 chr8 130545778 130546819 0.044 chr8 128745001 128752294 chr8 130546973 130552572 0.329 chr8 128745001 128752294 chr8 130593151 130597089 0 chr8 128745001 128752294 chr8 130598258 130600266 1 chr8 128745001 128752294 chr8 130690599 130698597 0.799 chr8 128745001 128752294 chr8 130698611 130700895 0.375 chr8 128745001 128752294 chr8 130708318 130711352 0.976 chr8 128745001 128752294 chr8 130737602 130739686 0.033 chr8 128745001 128752294 chr8 140642655 140643943 0 chr8 128752908 128753569 chr8 128979417 128982624 0 chr8 128752908 128753569 chr8 129201051 129204117 0 chr8 128755105 128756028 chr8 128772026 128773570 0 chr8 128755105 128756028 chr8 128922538 128924783 0.027 chr8 128755105 128756028 chr8 128926524 128927220 0 chr8 128755105 128756028 chr8 128982695 128983816 0 chr8 128772026 128773570 chr8 128830032 128831239 0.022 chr8 128772026 128773570 chr8 129060227 129063805 0 chr8 128772026 128773570 chr8 129334525 129335739 0 chr8 128812838 128813544 chr8 128973335 128974261 0 chr8 128830032 128831239 chr8 128906465 128907622 0 chr8 128830032 128831239 chr8 128979417 128982624 0 chr8 128830032 128831239 chr8 129209019 129210639 0 chr8 128830032 128831239 chr8 130690599 130698597 0.452 chr8 128863351 128864822 chr8 129201051 129204117 0 chr8 128871531 128872830 chr8 128906465 128907622 0 chr8 128871531 128872830 chr8 128922538 128924783 0 chr8 128871531 128872830 chr8 130546973 130552572 0 chr8 128871531 128872830 chr8 130598258 130600266 0 chr8 128896096 128897209 chr8 128910320 128912382 0.012 chr8 128896096 128897209 chr8 128971685 128973318 0 chr8 128896096 128897209 chr8 129665003 129666346 0 chr8 128906465 128907622 chr8 128910320 128912382 0.001 chr8 128906465 128907622 chr8 128971685 128973318 0 chr8 128906465 128907622 chr8 129056676 129058361 0.001 chr8 128906465 128907622 chr8 129334525 129335739 0 chr8 12890957 12892199 chr8 131354943 131355849 0 chr8 128910320 128912382 chr8 128922538 128924783 0.167 chr8 128910320 128912382 chr8 128926524 128927220 0.013 chr8 128910320 128912382 chr8 128961565 128962391 0 chr8 128910320 128912382 chr8 128971685 128973318 0 chr8 128910320 128912382 chr8 128979417 128982624 0 chr8 128910320 128912382 chr8 129060227 129063805 0.814 chr8 128910320 128912382 chr8 130546973 130552572 0 chr8 128910320 128912382 chr8 130568321 130570573 0.027 chr8 128922538 128924783 chr8 128926524 128927220 0.974 chr8 128922538 128924783 chr8 128971685 128973318 0 chr8 128922538 128924783 chr8 129005065 129006002 0 chr8 128922538 128924783 chr8 129060227 129063805 0 chr8 128922538 128924783 chr8 129209019 129210639 0 chr8 128922538 128924783 chr8 129334525 129335739 0.028 chr8 128926524 128927220 chr8 128979417 128982624 0 chr8 128926524 128927220 chr8 129026412 129027434 0 chr8 128926524 128927220 chr8 129077563 129078732 0 chr8 128958204 128958790 chr8 128971685 128973318 0 chr8 128961565 128962391 chr8 128971685 128973318 0 chr8 128971685 128973318 chr8 128973335 128974261 1 chr8 128971685 128973318 chr8 128979417 128982624 0 chr8 128971685 128973318 chr8 129056676 129058361 0 chr8 128971685 128973318 chr8 129060227 129063805 0.224 chr8 128971685 128973318 chr8 129209019 129210639 0 chr8 128973335 128974261 chr8 128979417 128982624 0.006 chr8 128973335 128974261 chr8 128982695 128983816 0.01 chr8 128973335 128974261 chr8 130708318 130711352 0 chr8 128979417 128982624 chr8 128982695 128983816 1 chr8 128979417 128982624 chr8 128989794 128990535 0 chr8 128979417 128982624 chr8 129026412 129027434 0 chr8 128979417 128982624 chr8 129056676 129058361 0.019 chr8 128979417 128982624 chr8 129058652 129059360 0 chr8 128979417 128982624 chr8 129060227 129063805 0 chr8 128979417 128982624 chr8 129089447 129090574 0 chr8 128979417 128982624 chr8 129201051 129204117 0.021 chr8 128982695 128983816 chr8 128988702 128989721 0 chr8 128982695 128983816 chr8 128989794 128990535 0 chr8 128982695 128983816 chr8 129026412 129027434 0 chr8 128982695 128983816 chr8 129056676 129058361 0 chr8 128982695 128983816 chr8 129060227 129063805 0 chr8 128988702 128989721 chr8 128989794 128990535 0.163 chr8 128988702 128989721 chr8 129060227 129063805 0 chr8 128988702 128989721 chr8 130698611 130700895 0 chr8 128988702 128989721 chr8 145800376 145801377 0 chr8 128989794 128990535 chr8 129060227 129063805 0 chr8 128989794 128990535 chr8 130598258 130600266 0.001 chr8 129026412 129027434 chr8 129060227 129063805 0.237 chr8 129026412 129027434 chr8 135702207 135704374 0 chr8 129056676 129058361 chr8 129058652 129059360 1 chr8 129056676 129058361 chr8 129060227 129063805 0.992 chr8 129056676 129058361 chr8 129074007 129074657 0 chr8 129056676 129058361 chr8 129188678 129190475 0 chr8 129056676 129058361 chr8 129201051 129204117 0.021 chr8 129058652 129059360 chr8 129060227 129063805 0.246 chr8 129060227 129063805 chr8 129074007 129074657 0 chr8 129060227 129063805 chr8 129188678 129190475 0 chr8 129060227 129063805 chr8 129201051 129204117 0 chr8 129060227 129063805 chr8 130546973 130552572 0 chr8 129074007 129074657 chr8 129077563 129078732 0.001 chr8 129074007 129074657 chr8 129188678 129190475 0.016 chr8 129077563 129078732 chr8 129201051 129204117 0 chr8 129139733 129140369 chr8 129200156 129200875 0 chr8 129139733 129140369 chr8 129201051 129204117 0 chr8 129179729 129180447 chr8 129201051 129204117 0 chr8 129179729 129180447 chr8 129209019 129210639 0 chr8 129188678 129190475 chr8 129196702 129197452 0 chr8 129188678 129190475 chr8 129334525 129335739 0 chr8 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We developed a novel analytical method to analyze ChIA-PET data that used a two-component Bayesian mixture model to accurately identify in vivo interactions from the ChIA-PET data by accurately estimating the difference between the biological signal and technical and biological noise by controlling for error within the ChIA-PET protocol and linear genomic distance. We defined an in vivo interaction as two regions of the genome brought together in the nucleus longer than expected at random given the linear genomic distance between those two region. Our intuition was that true in vivo interactions would follow one distribution where experimental noise would arise from a separate distribution, and these two groups could be learned from the data using a mixture model. After alignment, we defined a set of putative contacts where a putative contact was any two MACS1 peaks linked together by at least one mapped PET. The PET count for a putative interaction was the total number of unique PETs mapped at both ends of the putative contact. All putative contacts and their PET count were used in the estimation. We estimated the distributions two-component mixture model (described more below) from these putative contacts using origami-analysis. We specifically designed the model to have the second mixture component represent the distribution of the in vivo interactions, and we tested whether the estimated group means (described below) were significantly different to validate that model found at least two different groups. After the estimation of each component, for each putative contact we estimate the posterior probability of whether the putative contact was within the distribution of the second component. If this posterior probability was greater than 0.9, we called this putative contact an in vivo interaction. We used this threshold because it was a good balance between what be believed to be a high true positive rate while minimizing the false positive rate in each sample analyzed (although we believe that are a few in vivo interactions below this threshold as well). In general, we often displayed all putative contacts within the MYC TAD by this posterior probability, eliminating the need for a specific cutoff threshold.

For the analytical model, we wanted to build a model that was able to estimate and control for noise arising from the ChIA-PET protocol and linear genomic distance. The linear genomic distance is a potential source of noise in the data because regions of the genome closer together in linear genomic distance are on average more likely to have more frequent interactions by chance than regions of the genome farther away from each other, as observed in Hi-C data. We assume that these two sources of noise are independent Poisson processes from each other (since we are measuring the interaction frequency through PET counts), which appears to be a valid assumption in practice (Phanstiel et al., 2015). With this in mind, the parameters within the model were as follows:

P_(i)—the count of DNA mapped sequences/reads measured at position i, where i ∈{1 . . . N} and N is the total number of positions measured

Z_(i)—a latent variable having a value of either 0 or 1 measuring whether the measurement in P_(i) came from a technical artifact (0) or in vivo biology (1)

G_(ij)—a latent variable, where j ∈{0, 1}, measuring the number of counts observed for sample i if were part of component j

B_(ij)—a latent variable measuring the number of counts observed for sample i if were part of component j as a function of the bias due to the genomic distance that sample i spans in the genome (where the distance is assumed to be a constant d_(i) for that sample i)

R_(ij)—a latent variable measuring sum of the two independent processes G_(ij) and B_(ij) for sample i if were part of component j

λ_(j)—a parameter describing the mean of latent variable G_(.j) for all samples, and we guarantee that λ₁>λ₀ to maintain identifiability of each component

v_(i) (d)—a parameter describing the mean of the latent variable B_(.j) at distance d

w_(ij)—a parameter describing the binomial probability that sample I is part of component j

a_(i), b_(i)—a set of constants on the prior distribution of w_(i1) to adjust our prior belief in sample i based on our understanding of the biology we have already validated in lab

More specifically, the model is parametrized as follows:

λ_(j)∼Gamma(1, 1) w_(i 1)∼Beta(a_(i), b_(i)) G_(j)λ_(j)∼Poisson(λ_(j)) B_(ij)λ_(j), d_(i)∼Poisson(v_(j)d_(i)) R_(ij) = G_(ij) + B_(ij)λ_(j), v_(j), d_(i)∼Poisson(λ_(j) + v_(j)d_(i)) ${P_{i}R_{ij}},\lambda_{j},v_{j},{d_{i} = {\sum\limits_{j \in {\{{0,1}\}}}\; {w_{ij}*R_{j}}}}$ P_(i)Z_(i) = z_(i), λ_(z_(i)), v_(z_(i))d_(i)∼Poisson(λ_(j) + v_(j)d_(i)) w_(i 1)Z_(i) = z_(i), P_(i)∼Beta(a_(i) + z_(i), b_(i) + (1 − z_(i)))  And w_(i 0) = 1 − w_(i 1).

The distribution of the parameters and hyperparameters were simulated by Markov Chain Monte Carlo (MCMC) using either Gibbs sampling or the Metropolis-Hastings algorithm as appropriate. To speed up the simulation between G_(.j) and B_(ij), the G_(.j) parameter is updated first. Then the B_(ij) parameters is updated using the difference between the PETs for G_(.j) and the number of PETs observed for each contact according to the component they are assigned to in that iteration. Additionally, the mean of G₀ is enforced to be less than the mean of G₁, although in practice the mean of G₁ was always strictly greater than the mean of G₀ during the MCMC run so this was never a problem.

The parameter v_(j) (d) is the mean of the Poisson process estimating the biological bias from the linear genomic distance between the two ends of the putative contact as a function of this distance. To simplify processing, we estimated this function at each iterative using a smoothed cubic spline regression for putative contacts within group j. This approximation worked well by generating trends consistent with the power-law decay observed in Hi-C data sets.

The priors a_(.i) and b_(.i) are set to be minimally informative as possible. The a_(.i) hyperparameter is the frequency of the number of contacts sharing one of the same anchors that have a strictly lower measured PET count than the putative contact i. The b_(.i) hyperparamer is set to be the frequency of putative contacts sharing the same anchor that have strictly higher number of observed PETs linking the anchors plus the ratio of the multiplication of the depth of reads at both anchors of the putative contact divided by the median depth across all putative contacts floored at 0. We found setting the priors with a non-informative Beta distribution (i.e., Beta(1,1)) would also generally call the same in vivo interactions but call many more interactions from the putative contacts, where we believed many more of these were artifacts. Hence, we found this minimally informative prior to be more useful for us biologically.

Each run of origami-analysis was for 1,000 iterations with a 100 step burn-in period. We chose this number of iterations because the model tended to converge fairly quickly given the complexity of these ChIA-PET data sets. The output of origami is the estimated posterior probability that the putative contact arose from the distribution estimated for the second mixture component, which is assumed to model in vivo interactions within the ChIA-PET experiment. Accordingly, putative contact with a posterior probability closer to 1 are believed to be more likely to be in vivo interactions.

CTCF Motif Analysis

CTCF motifs were called in the human and mouse genomes (using hg19 and mm9 assemblies, respectively) using fimo (Grant et al., 2011). The CTCF motif from the JASPAR CORE 2014 database was used. The fimo p-value threshold was set to 1e-2 and the max-stored-scores parameter set to 100000000. To rank the importance of individual CTCF motifs, the motifs within the targeted CTCF peak upstream of MYC were ranked by their score within the score column in the GFF output of fimo.

Identification of Genes with a Putative Enhancer Docking Site

Genes with a putative enhancer docking site were identified by filtering the list of all 26,801 annotated genes down to those which occurred within a constitutive insulated neighborhood, had a constitutive CTCF site within 2.5 kb of the TSS, and exhibited differential enhancer usage across the cell lines HCT-116, Jurkat, and K562. Constitutive insulated neighborhoods were identified as follows. First, CTCF binding sites and cohesin binding sites were identified in HCT-116, Jurkat, and K562 cells. Cohesin ChIA-PET in the three cell types were processed with the Oragami pipeline as described below, and two CTCF bound sites that are connected by a cohesin ChIA-PET interaction were annotated as CTCF-CTCF/cohesin interactions in each cell type (i.e. insulated neighborhoods). Insulated neighborhoods were scored as constitutive across two cell types if they had a reciprocal overlap of at least 80% of the length of the interaction. The ChIA-PET datasets are likely not saturated, suggesting that not every interaction found within a cell will be potentially represented in the dataset. Therefore, we defined constitutive insulated neighborhoods as the set of insulated neighborhoods from all three cell types that were found overlapping in at least two of the three cell types. Conserved CTCF sites at gene's TSSs were defined as those sites that occur within 2.5 kb of the TSS and overlap by at least lbp across all 3 cell types. A gene was considered to use differential enhancers if in one of the cell types there was an enhancer present within the confines of the gene's constitutive insulated neighborhood that was not present in at least one of the other two cell types. Applying these three filters results in 1,725 genes that may utilize an enhancer docking site.

Data and Software Availability

ORIGAMI: https://github.com/younglab/origami using version alpha20160828.

The ChIP-seq data have been deposited in the Gene Expression Omnibus (GEO) under ID code GSE92879

The HiChIP data have been deposited in the Gene Expression Omnibus (GEO) under ID codes GSE92881

The 4C-seq data have been deposited in the the Gene Expression Omnibus (GEO) under ID code GSE92880 Software and Algorithms

Origami This study/Weintraub https://github.com/younglab/origami et al ROSE Whyte et al https://bitbucket.org/young_computation/rose 4C Fourfold This study https://github.com/younglab/fourfold BISMARK Krueger et al. https://github.com/FelixKrueger/Bismark Bowtie Langmead et al., 2009 http://bowtiebio.sourceforge.net/index.shtml Samtools Li et al., 2009 http://samtools.sourceforge.net MACS Zhang et al., 2008 http://liulab.dfci.harvard.edu/MACS/index.html BEDTools Quinlan et al., 2010 http://bedtools.readthedocs.io UCSC Genome Browser Kent et al., 2002 http://genome.ucsc.edu/cgibin/hgGateway WASHU EpiGenome browser Zhou et al., 2011 http://epigenomegateway.wustl.edu/ 3D Genome viewer Wang et al., 2017 http://www.3dgenome.org Kallisto Bray et al., 2016 https://pachterlab.github.io/kallisto/

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1. A composition comprising a. a catalytically inactive site specific nuclease fused to an effector domain having methylation activity or one or more nucleic acids encoding the same; and b. one or more guide sequences homologous to at least a portion of promoter region CTCF binding site of a gene or one or more nucleic acids encoding one or more guide sequences homologous to at least a portion of the promoter region CTCF binding site.
 2. The composition of claim 1, wherein the gene is selected from an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S
 1. 3.-7. (canceled)
 8. The composition of claim 1, wherein the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DMNT3A-3L or dCas9-DMNT3A-3L without a 5′ NLS. 9.-11. (canceled)
 12. The composition of claim 1, wherein the one or more guide sequences comprise a sequence homologous to a sequence selected from the group consisting of SEQ ID NOS. 1-8.
 13. The composition of any one of claim 1, wherein the composition is for treating cancer. 14.-26. (canceled)
 27. A method for methylating a promoter region CTCF binding site of a gene in a cell comprising introducing into the cell: a. a catalytically inactive site specific nuclease fused to an effector domain having methylation activity; and b. one or more guide sequences homologous to at least a portion of the promoter region CTCF binding site, thereby methylating the promoter region CTCF binding site.
 28. The method of claim 27, wherein the gene is selected from an oncogene, MYC, TG1F1, VEGFA1, RUNX1, CDK6, BCL2L1, PIM1, CSNK1A1 or a gene listed in Table S1. 29.-32. (canceled)
 33. The method of claim 27, wherein the effector domain having methylation activity is DMNT3A-3L.
 34. The method of claim 27, wherein the catalytically inactive site specific nuclease fused to an effector domain having methylation activity is dCas9-DMNT3A-3L or dCas9-DMNT3A-3L without a 5′ NLS. 35.-37. (canceled)
 38. The method of claim 27, wherein the one or more guide sequences comprise a sequence encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOS. 1-8.
 39. (canceled)
 40. The method of claim 27, wherein the cell is a human cell.
 41. The method of claim 27, wherein the cell is contacted in vivo in a subject a mouse cell.
 42. The method of claim 27, wherein the cell is a cancer cell or a pre-cancerous cell.
 43. The method of claim 42, wherein the cancer cell is a colorectal cancer cell, a leukemia cell, or a breast cancer cell.
 44. The method of claim 27, wherein the expression of the gene in the cell is reduced.
 45. The method of claim 27, wherein the expression of mRNA of the gene is reduced by about 50% or more. 46.-63. (canceled)
 64. The method of claim 41, wherein the subject has cancer and reducing the expression of the gene in the subject treats the cancer.
 65. The method of claim 64, wherein the gene is MYC.
 66. The method of claim 64, wherein the cancer is colorectal cancer, leukemia, or breast cancer. 67.-88. (canceled)
 89. A method of screening for an anti-cancer compound comprising, a. contacting a cell with a test agent; and b. measuring methylation in a promoter region CTCF binding site of a gene, wherein the test agent is identified as a candidate anti-cancer compound if the level of methylation of the promoter region CTCF binding site in the cell contacted with the test agent is modulated as compared to the level of methylation of said promoter region CTCF binding site in a control cell not contacted with the test agent. 90.-95. (canceled) 